Rational design of structure-based vaccines targeting misfolded alpha-synuclein conformers of Parkinson's disease and related disorders.

Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are neurodegenerative disorders caused by the accumulation of misfolded alpha-synuclein protein. Developing effective vaccines against synucleinopathies is challenging due to the difficulty of stimulating an immune-specific response against alpha-synuclein without causing harmful autoimmune reactions, selectively targeting only pathological forms of alpha-synuclein. Previous attempts using linear peptides and epitopes without control of the antigen structure failed in clinical trials. The immune system was unable to distinguish between native alpha-synuclein and its amyloid form. The prion domain of the fungal HET-s protein was selected as a scaffold to introduce select epitopes from the surface of alpha-synuclein fibrils. Four vaccine candidates were generated by introducing specific amino acid substitutions onto the surface of the scaffold protein. The approach successfully mimicked the stacking of the parallel in-register beta-sheet structure seen in alpha-synuclein fibrils. All vaccine candidates induced substantial levels of IgG antibodies that recognized pathological alpha-synuclein fibrils derived from a synucleinopathy mouse model. Furthermore, the antisera recognized pathological alpha-synuclein aggregates in brain lysates from patients who died from DLB, MSA, or PD, but did not recognize linear alpha-synuclein peptides. Our approach, based on the rational design of vaccines using the structure of alpha-synuclein amyloid fibrils and strict control over the exposed antigen structure used for immunization, as well as the ability to mimic aggregated alpha-synuclein, provides a promising avenue toward developing effective vaccines against alpha-synuclein fibrils.

Ensuring sexual and reproductive healthcare services amidst a pandemic: Experiences from health workers in Lima, Peru.

Nowadays there is an emerging interest on health system resilience capacity during emergencies as the one created by the COVID-19 Pandemic. This article contributes to this emerging field of studies by analysing the impact of the state´s policy responses COVID-19 (as lockdowns) on the Peruvian health system, specifically on the delivery of non-covid services, sexual and reproductive health services, and describe the strategies deployed by health workers to adapt to the COVID-19 crisis in Peru, a country that have been dramatically impacted by the pandemic. The article, based on the analysis of depth interviews with 11 health workers and one health supervisor working at sexual and reproductive health services at public health services Lima during 2020 and 2021, describe how pre-existing conditions of the health system (as poor infrastructure and deficit of human resources) magnified the negative effects of the measures taken to control de pandemic, undermining the "resilience" of the health system.

Alzheimer's disease associated isoforms of human CD33 distinctively modulate microglial cell responses in 5XFAD mice.

Microglia play diverse pathophysiological roles in Alzheimer's disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans. We address this challenge by studying transgenic mice expressing either of the human CD33 isoforms crossed with the 5XFAD mouse model of amyloidosis and find that human CD33 isoforms have opposing effects on the response of microglia to amyloid-ß (Aß) deposition. Mice expressing CD33M have increased Aß levels, more diffuse plaques, fewer disease-associated microglia, and more dystrophic neurites compared to 5XFAD control mice. Conversely, CD33m promotes plaque compaction and microglia-plaque contacts, and minimizes neuritic plaque pathology, highlighting an AD protective role for this isoform. Protective phenotypes driven by CD33m are detected at an earlier timepoint compared to the more aggressive pathology in CD33M mice that appears at a later timepoint, suggesting that CD33m has a more prominent impact on microglia cell function at earlier stages of disease progression. In addition to divergent roles in modulating phagocytosis, scRNAseq and proteomics analyses demonstrate that CD33m+ microglia upregulate nestin, an intermediate filament involved in cell migration, at plaque contact sites. Overall, our work provides new functional insights into how CD33, as a top genetic susceptibility factor for AD, modulates microglial cell function.

In Vitro and In Vivo Evidence towards Fibronectin's Protective Effects against Prion Infection.

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.

[Responding to the sexual and reproductive health needs of Venezuelan migrant women in LimaResposta às necessidades de saúde sexual e reprodutiva de migrantes venezuelanas em Lima]. / Respondiendo a las necesidades de salud sexual y reproductiva de las migrantes venezolanas en Lima.

Objectives: To present and analyze the Peruvian health system's response to the sexual and reproductive health needs of Venezuelan women living in the city of Lima, Peru, and to identify some of the reasons underlying this response. Methods: Information was collected through semi-structured, in-depth telephone interviews with 30 Venezuelan women, 10 healthcare workers, and two Ministry of Health officials. Results: Based on the experiences of Venezuelan women who sought care through these services during 2019-2020 and the perspectives of healthcare personnel and health authorities, we present an analysis of the public health services' capacity and limitations in meeting the sexual and reproductive health needs of this population. Migrant women's testimonies reported a positive experience with a health system that, despite shortcomings, responds to the most common sexual and reproductive health needs. These perspectives parallel the testimonies of healthcare personnel and authorities who emphasized the existence of priority policies for sexual and reproductive health care. Conclusion: This study shows how a national priority framework (reducing maternal mortality), accompanied by operational mechanisms for social protection (such as the Comprehensive Health Insurance program), represent complementary instruments that have a positive impact on and extend benefits to migrants, even though this population was not considered when designing these policies.

Objetivo: Apresentar e analisar a resposta do sistema de saúde peruano às necessidades de saúde sexual e reprodutiva de mulheres venezuelanas radicadas em Lima, Peru, e identificar algumas explicações para essa resposta. Métodos: Entrevistas telefônicas semiestruturadas detalhadas com 30 mulheres venezuelanas, 10 profissionais de saúde e 2 funcionários do Ministério da Saúde. Resultados: Com base nas experiências das mulheres venezuelanas que recorreram a esses serviços no período de 2019 a 2020 e nas perspectivas de profissionais e autoridades de saúde, apresentamos uma análise da capacidade e das limitações dos serviços de saúde pública para atender às necessidades de saúde sexual e reprodutiva dessa população. Os relatos das mulheres migrantes indicam uma experiência positiva com um sistema de saúde, que, apesar das deficiências, responde às necessidades mais comuns de saúde sexual e reprodutiva. Isso está em conformidade com os relatos dos profissionais de saúde e das autoridades, que enfatizam a existência de políticas prioritárias de atenção à saúde sexual e reprodutiva. Conclusão: Este estudo mostra de que maneira um âmbito de prioridade nacional (reduzir a mortalidade materna) e mecanismos operacionais de proteção social (como o Seguro Integral de Saúde) se convertem em instrumentos complementares, afetando positivamente e estendendo benefícios à população migrante, embora essa população não tenha sido levada em consideração quando da elaboração dessas políticas.

Respondiendo a las necesidades de salud sexual y reproductiva de las migrantes venezolanas en Lima

[RESUMEN]. Objetivo. Presentar y analizar la respuesta que el sistema de salud peruano viene dando a las necesidades en salud sexual y reproductiva de las mujeres venezolanas que radican en la ciudad de Lima, Perú e identificar algunas de las razones que nos permite entender esta respuesta. Métodos. La información se recogió mediante entrevistas a profundidad semiestructuradas por vía telefónica a 30 mujeres venezolanas, 10 trabajadores de salud y 2 funcionarios del Ministerio de Salud. Resultados. A partir de las experiencias de mujeres venezolanas que acudieron a estos servicios durante el 2019-2020 y de las perspectivas del personal y autoridades de salud presentamos un análisis de la capacidad y limitaciones que los servicios de salud públicos tienen para atender las necesidades de salud sexual y reproductiva de esta población. Los testimonios de las mujeres migrantes reportan una experiencia positiva con un sistema de salud que, a pesar de las deficiencias, responde a las necesidades de salud sexual y reproductiva más comunes. Estas coinciden con los testimonios del personal de salud y con las de las autoridades quienes enfatizan la existencia de políticas prioritarias para la atención de la Salud Sexual y Reproductiva. Conclusión. Este estudio muestra cómo un marco de prioridad nacional (disminuir la mortalidad materna), acompañado de mecanismos operativos de protección social (como el Seguro Integral de Salud), se convierten en instrumentos complementarios, que repercute de manera positiva y extiende beneficios para las y los migrantes, a pesar de no haber considerado a esta población durante el diseño de estas políticas.

[ABSTRACT]. Objectives. To present and analyze the Peruvian health system's response to the sexual and reproductive health needs of Venezuelan women living in the city of Lima, Peru, and to identify some of the reasons under- lying this response. Methods. Information was collected through semi-structured, in-depth telephone interviews with 30 Venezuelan women, 10 healthcare workers, and two Ministry of Health officials. Results. Based on the experiences of Venezuelan women who sought care through these services during 2019-2020 and the perspectives of healthcare personnel and health authorities, we present an analysis of the public health services' capacity and limitations in meeting the sexual and reproductive health needs of this population. Migrant women's testimonies reported a positive experience with a health system that, despite shortcomings, responds to the most common sexual and reproductive health needs. These perspectives parallel the testimonies of healthcare personnel and authorities who emphasized the existence of priority policies for sexual and reproductive health care. Conclusion. This study shows how a national priority framework (reducing maternal mortality), accompanied by operational mechanisms for social protection (such as the Comprehensive Health Insurance program), represent complementary instruments that have a positive impact on and extend benefits to migrants, even though this population was not considered when designing these policies.

[RESUMO]. Objetivo. Apresentar e analisar a resposta do sistema de saúde peruano às necessidades de saúde sexual e reprodutiva de mulheres venezuelanas radicadas em Lima, Peru, e identificar algumas explicações para essa resposta. Métodos. Entrevistas telefônicas semiestruturadas detalhadas com 30 mulheres venezuelanas, 10 profissionais de saúde e 2 funcionários do Ministério da Saúde. Resultados. Com base nas experiências das mulheres venezuelanas que recorreram a esses serviços no período de 2019 a 2020 e nas perspectivas de profissionais e autoridades de saúde, apresentamos uma análise da capacidade e das limitações dos serviços de saúde pública para atender às necessidades de saúde sexual e reprodutiva dessa população. Os relatos das mulheres migrantes indicam uma experiência positiva com um sistema de saúde, que, apesar das deficiências, responde às necessidades mais comuns de saúde sexual e reprodutiva. Isso está em conformidade com os relatos dos profissionais de saúde e das autoridades, que enfatizam a existência de políticas prioritárias de atenção à saúde sexual e reprodutiva. Conclusão. Este estudo mostra de que maneira um âmbito de prioridade nacional (reduzir a mortalidade materna) e mecanismos operacionais de proteção social (como o Seguro Integral de Saúde) se convertem em instrumentos complementares, afetando positivamente e estendendo benefícios à população migrante, embora essa população não tenha sido levada em consideração quando da elaboração dessas políticas.

Respondiendo a las necesidades de salud sexual y reproductiva de las migrantes venezolanas en Lima / Responding to the sexual and reproductive health needs of Venezuelan migrant women in Lima / Resposta às necessidades de saúde sexual e reprodutiva de migrantes venezuelanas em Lima

RESUMEN Objetivo. Presentar y analizar la respuesta que el sistema de salud peruano viene dando a las necesidades en salud sexual y reproductiva de las mujeres venezolanas que radican en la ciudad de Lima, Perú e identificar algunas de las razones que nos permite entender esta respuesta. Métodos. La información se recogió mediante entrevistas a profundidad semiestructuradas por vía telefónica a 30 mujeres venezolanas, 10 trabajadores de salud y 2 funcionarios del Ministerio de Salud. Resultados. A partir de las experiencias de mujeres venezolanas que acudieron a estos servicios durante el 2019-2020 y de las perspectivas del personal y autoridades de salud presentamos un análisis de la capacidad y limitaciones que los servicios de salud públicos tienen para atender las necesidades de salud sexual y reproductiva de esta población. Los testimonios de las mujeres migrantes reportan una experiencia positiva con un sistema de salud que, a pesar de las deficiencias, responde a las necesidades de salud sexual y reproductiva más comunes. Estas coinciden con los testimonios del personal de salud y con las de las autoridades quienes enfatizan la existencia de políticas prioritarias para la atención de la Salud Sexual y Reproductiva. Conclusión. Este estudio muestra cómo un marco de prioridad nacional (disminuir la mortalidad materna), acompañado de mecanismos operativos de protección social (como el Seguro Integral de Salud), se convierten en instrumentos complementarios, que repercute de manera positiva y extiende beneficios para las y los migrantes, a pesar de no haber considerado a esta población durante el diseño de estas políticas.

ABSTRACT Objectives. To present and analyze the Peruvian health system's response to the sexual and reproductive health needs of Venezuelan women living in the city of Lima, Peru, and to identify some of the reasons underlying this response. Methods. Information was collected through semi-structured, in-depth telephone interviews with 30 Venezuelan women, 10 healthcare workers, and two Ministry of Health officials. Results. Based on the experiences of Venezuelan women who sought care through these services during 2019-2020 and the perspectives of healthcare personnel and health authorities, we present an analysis of the public health services' capacity and limitations in meeting the sexual and reproductive health needs of this population. Migrant women's testimonies reported a positive experience with a health system that, despite shortcomings, responds to the most common sexual and reproductive health needs. These perspectives parallel the testimonies of healthcare personnel and authorities who emphasized the existence of priority policies for sexual and reproductive health care. Conclusion. This study shows how a national priority framework (reducing maternal mortality), accompanied by operational mechanisms for social protection (such as the Comprehensive Health Insurance program), represent complementary instruments that have a positive impact on and extend benefits to migrants, even though this population was not considered when designing these policies.

RESUMO Objetivo. Apresentar e analisar a resposta do sistema de saúde peruano às necessidades de saúde sexual e reprodutiva de mulheres venezuelanas radicadas em Lima, Peru, e identificar algumas explicações para essa resposta. Métodos. Entrevistas telefônicas semiestruturadas detalhadas com 30 mulheres venezuelanas, 10 profissionais de saúde e 2 funcionários do Ministério da Saúde. Resultados. Com base nas experiências das mulheres venezuelanas que recorreram a esses serviços no período de 2019 a 2020 e nas perspectivas de profissionais e autoridades de saúde, apresentamos uma análise da capacidade e das limitações dos serviços de saúde pública para atender às necessidades de saúde sexual e reprodutiva dessa população. Os relatos das mulheres migrantes indicam uma experiência positiva com um sistema de saúde, que, apesar das deficiências, responde às necessidades mais comuns de saúde sexual e reprodutiva. Isso está em conformidade com os relatos dos profissionais de saúde e das autoridades, que enfatizam a existência de políticas prioritárias de atenção à saúde sexual e reprodutiva. Conclusão. Este estudo mostra de que maneira um âmbito de prioridade nacional (reduzir a mortalidade materna) e mecanismos operacionais de proteção social (como o Seguro Integral de Saúde) se convertem em instrumentos complementares, afetando positivamente e estendendo benefícios à população migrante, embora essa população não tenha sido levada em consideração quando da elaboração dessas políticas.

Probing the origin of prion protein misfolding via reconstruction of ancestral proteins.

Prion diseases are fatal neurodegenerative diseases caused by pathogenic misfolding of the prion protein, PrP. They are transmissible between hosts, and sometimes between different species, as with transmission of bovine spongiform encephalopathy to humans. Although PrP is found in a wide range of vertebrates, prion diseases are seen only in certain mammals, suggesting that infectious misfolding was a recent evolutionary development. To explore when PrP acquired the ability to misfold infectiously, we reconstructed the sequences of ancestral versions of PrP from the last common primate, primate-rodent, artiodactyl, placental, bird, and amniote. Recombinant ancestral PrPs were then tested for their ability to form ß-sheet aggregates, either spontaneously or when seeded with infectious prion strains from human, cervid, or rodent species. The ability to aggregate developed after the oldest ancestor (last common amniote), and aggregation capabilities diverged along evolutionary pathways consistent with modern-day susceptibilities. Ancestral bird PrP could not be seeded with modern-day prions, just as modern-day birds are resistant to prion disease. Computational modeling of structures suggested that differences in helix 2 could account for the resistance of ancestral bird PrP to seeding. Interestingly, ancestral primate PrP could be converted by all prion seeds, including both human and cervid prions, raising the possibility that species descended from an ancestral primate have retained the susceptibility to conversion by cervid prions. More generally, the results suggest that susceptibility to prion disease emerged prior to ~100 million years ago, with placental mammals possibly being generally susceptible to disease.

Distinctive Toll-like Receptors Gene Expression and Glial Response in Different Brain Regions of Natural Scrapie.

Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumulation of disease-specific prion protein (PrPSc), spongiform changes, neuronal loss, and gliosis. Growing evidence shows that the neuroinflammatory response is a key component of prion diseases and contributes to neurodegeneration. Toll-like receptors (TLRs) have been proposed as important mediators of innate immune responses triggered in the central nervous system in other human neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. However, little is known about the role of TLRs in prion diseases, and their involvement in the neuropathology of natural scrapie has not been studied. We assessed the gene expression of ovine TLRs in four anatomically distinct brain regions in natural scrapie-infected sheep and evaluated the possible correlations between gene expression and the pathological hallmarks of prion disease. We observed significant changes in TLR expression in scrapie-infected sheep that correlate with the degree of spongiosis, PrPSc deposition, and gliosis in each of the regions studied. Remarkably, TLR4 was the only gene upregulated in all regions, regardless of the severity of neuropathology. In the hippocampus, we observed milder neuropathology associated with a distinct TLR gene expression profile and the presence of a peculiar microglial morphology, called rod microglia, described here for the first time in the brain of scrapie-infected sheep. The concurrence of these features suggests partial neuroprotection of the hippocampus. Finally, a comparison of the findings in naturallyinfected sheep versus an ovinized mouse model (tg338 mice) revealed distinct patterns of TLRgene expression.

Implications of exosomes derived from cholesterol-accumulated astrocytes in Alzheimer's disease pathology.

Amyloid ß (Aß) peptides generated from the amyloid precursor protein (APP) play a critical role in the development of Alzheimer's disease (AD) pathology. Aß-containing neuronal exosomes, which represent a novel form of intercellular communication, have been shown to influence the function/vulnerability of neurons in AD. Unlike neurons, the significance of exosomes derived from astrocytes remains unclear. In this study, we evaluated the significance of exosomes derived from U18666A-induced cholesterol-accumulated astrocytes in the development of AD pathology. Our results show that cholesterol accumulation decreases exosome secretion, whereas lowering cholesterol increases exosome secretion, from cultured astrocytes. Interestingly, exosomes secreted from U18666A-treated astrocytes contain higher levels of APP, APP-C-terminal fragments, soluble APP, APP secretases and Aß1-40 than exosomes secreted from control astrocytes. Furthermore, we show that exosomes derived from U18666A-treated astrocytes can lead to neurodegeneration, which is attenuated by decreasing Aß production or by neutralizing exosomal Aß peptide with an anti-Aß antibody. These results, taken together, suggest that exosomes derived from cholesterol-accumulated astrocytes can play an important role in trafficking APP/Aß peptides and influencing neuronal viability in the affected regions of the AD brain.

Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm.

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.

Quaternary Structure Changes for PrPSc Predate PrPC Downregulation and Neuronal Death During Progression of Experimental Scrapie Disease.

Prion diseases are fatal neurodegenerative diseases in mammals with the unique characteristics of misfolding and aggregation of the cellular prion protein (PrPC) to the scrapie prion (PrPSc). Although neuroinflammation and neuronal loss feature within the disease process, the details of PrPC/PrPSc molecular transition to generate different aggregated species, and the correlation between each species and sequence of cellular events in disease pathogenesis are not fully understood. In this study, using mice inoculated with the RML isolate of mouse-adapted scrapie as a model, we applied asymmetric flow field-flow fractionation to monitor PrPC and PrPSc particle sizes and we also measured seeding activity and resistance to proteases. For cellular analysis in brain tissue, we measured inflammatory markers and synaptic damage, and used the isotropic fractionator to measure neuronal loss; these techniques were applied at different timepoints in a cross-sectional study of disease progression. Our analyses align with previous reports defining significant decreases in PrPC levels at pre-clinical stages of the disease and demonstrate that these decreases become significant before neuronal loss. We also identified the earliest PrPSc assemblies at a timepoint equivalent to 40% elapsed time for the disease incubation period; we propose that these assemblies, mostly composed of proteinase K (PK)-sensitive species, play an important role in triggering disease pathogenesis. Lastly, we show that the PK-resistant assemblies of PrPSc that appear at timepoints close to the terminal stage have similar biophysical characteristics, and hence that preparative use of PK-digestion selects for this specific subpopulation. In sum, our data argue that qualitative, as well as quantitative, changes in PrP conformers occur at the midpoint of subclinical phase; these changes affect quaternary structure and may occur at the threshold where adaptive responses become inadequate to deal with pathogenic processes.

Native nanodiscs formed by styrene maleic acid copolymer derivatives help recover infectious prion multimers bound to brain-derived lipids.

Prions are lipidated proteins that interact with endogenous lipids and metal ions. They also assemble into multimers and propagate into the infectious scrapie form known as PrPSc The high-resolution structure of the infectious PrPSc state remains unknown, and its analysis largely relies on detergent-based preparations devoid of endogenous ligands. Here we designed polymers that allow isolation of endogenous membrane:protein assemblies in native nanodiscs without exposure to conventional detergents that destabilize protein structures and induce fibrillization. A set of styrene-maleic acid (SMA) polymers including a methylamine derivative facilitated gentle release of the infectious complexes for resolution of multimers, and a thiol-containing version promoted crystallization. Polymer extraction from brain homogenates from Syrian hamsters infected with Hyper prions and WT mice infected with Rocky Mountain Laboratories prions yielded infectious prion nanoparticles including oligomers and microfilaments bound to lipid vesicles. Lipid analysis revealed the brain phospholipids that associate with prion protofilaments, as well as those that are specifically enriched in prion assemblies captured by the methylamine-modified copolymer. A comparison of the infectivity of PrPSc attached to SMA lipid particles in mice and hamsters indicated that these amphipathic polymers offer a valuable tool for high-yield production of intact, detergent-free prions that retain in vivo activity. This native prion isolation method provides an avenue for producing relevant prion:lipid targets and potentially other proteins that form multimeric assemblies and fibrils on membranes.

Early stages of aggregation of engineered α-synuclein monomers and oligomers in solution.

α-Synuclein is a protein that aggregates as amyloid fibrils in the brains of patients with Parkinson's disease and dementia with Lewy bodies. Small oligomers of α-synuclein are neurotoxic and are thought to be closely associated with disease. Whereas α-synuclein fibrillization and fibril morphologies have been studied extensively with various methods, the earliest stages of aggregation and the properties of oligomeric intermediates are less well understood because few methods are able to detect and characterize early-stage aggregates. We used fluorescence spectroscopy to investigate the early stages of aggregation by studying pairwise interactions between α-synuclein monomers, as well as between engineered tandem oligomers of various sizes (dimers, tetramers, and octamers). The hydrodynamic radii of these engineered α-synuclein species were first determined by fluorescence correlation spectroscopy and dynamic light scattering. The rate of pairwise aggregation between different species was then monitored using dual-color fluorescence cross-correlation spectroscopy, measuring the extent of association between species labelled with different dyes at various time points during the early aggregation process. The aggregation rate and extent increased with tandem oligomer size. Self-association of the tandem oligomers was found to be the preferred pathway to form larger aggregates: interactions between oligomers occurred faster and to a greater extent than interactions between oligomers and monomers, indicating that the oligomers were not as efficient in seeding further aggregation by addition of monomers. These results suggest that oligomer-oligomer interactions may play an important role in driving aggregation during its early stages.

Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein.

The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

Toll-like receptor-mediated immune response inhibits prion propagation.

Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion diseases is neuroinflammation characterized by activation of neuroglia surrounding prion deposition. The cause and effect of this cellular response, however, is unclear. We investigated innate immune defenses against prion infection using primary mixed neuronal and glial cultures. Conditional prion propagation occurred in glial cultures depending on their immune status. Preconditioning of the cells with the toll-like receptor (TLR) ligand, lipopolysaccharide, resulted in a reduction in prion propagation, whereas suppression of the immune responses with the synthetic glucocorticoid, dexamethasone, increased prion propagation. In response to recombinant prion fibrils, glial cells up-regulated TLRs (TLR1 and TLR2) expression and secreted cytokines (tumor necrosis factor-α, interleukin-1ß, interleukin-6, granulocyte-macrophage colony-stimulating factor, and interferon-ß). Preconditioning of neuronal and glial cultures with recombinant prion fibrils inhibited prion replication and altered microglial and astrocytic populations. Our results provide evidence that, in early stages of prion infection, glial cells respond to prion infection through TLR-mediated innate immunity.

Enfoque basado en riesgo desde la etapa de desarrollo del producto succinilcolina infantil 100 mg / Risk-based approach from the development phase of 100mg infant succinylcoline product

Introducción: en la actualidad las guías emitidas por la Agencia de Medicamentos y Alimentos, de los EU considera que los procesos fallan porque las fuentes no son debidamente identificadas, eliminadas o controladas y plantean un nuevo enfoque de Buenas Prácticas de Producción basado en riesgos. Objetivo: aplicar un enfoque basado en riesgos en el aseguramiento de la calidad desde la etapa de desarrollo del producto succinilcolina 100 mg. Métodos: se realizó una breve descripción del proceso mediante un diagrama de bloque, se empleó el Árbol de Fallas como herramienta para identificar las posibles fuentes de falla; la valoración de riesgos partiendo de la identificación de las posibles fuentes de fallos se ejecutó a través de un método matricial. Se identificó un orden de prioridad en la toma de acciones correctivas para eliminar o mitigar el riesgo de ocurrencia. Resultados: el diagrama de bloque permitió tener un conocimiento sobre el proceso, lo que contribuyó de forma decisiva a un mejor ejercicio de la gestión de riesgos. El Árbol de Fallas resultó útil al revelar de forma gráfica las diferentes combinaciones de fallos e interrelaciones entre causa y efecto que pudieran dar lugar al evento tope indeseado. El método empleado para la valoración de riesgos permitió determinar las prioridades, siendo el tiempo extensivo de llenado (fuera de 2-8 °C) el componente crítico a considerar con especial atención; si se tiene en cuenta que la estabilidad de este producto se puede afectar a temperaturas superiores a estas, por lo que disponer de un plan de acciones correctivas acorde a los riesgos identificados permitirá la eliminación o mitigación de las mismas. Conclusiones: la herramienta de gestión de riesgos permitió identificar desde la etapa de desarrollo del producto succinilcolina 100 mg, las principales fuentes de fallas relacionadas con este proceso(AU)

Introduction: the guidelines presently issued by the Food and Drug Agency of the United States considers that processes fail because the sources are not duly identified, eliminated or controlled and submit a new approach of Good Manufacture Practice based on risks. Objective: to apply a risk-based approach to the quality assurance from the development phase of the 100 mg succinylcoline product. Methods: the process was briefly described through a block diagram with Failure Tree as a tool for identification of possible sources of failures; the risk assessment based on the detection of the possible sources of failures was made with the matrix method. An order of priority was given in the implementation of corrective actions to eliminate or mitigate the risk of occurrence. Results: the block diagram allowed knowing the process, which contributed in a decisive way to a better application of risk management. The Failure Tree proved to be useful when showing in a graphical way the different failure combinations and interrelations between cause and effect that might give rise to the unwanted top event. The method for the risk assessment made it possible to determine priorities, being the long time of filling (not within 2 to 8oC) the critical component to be specially considered if one takes into account that stability of the product may be affected by higher temperatures, therefore, a plan of corrective actions according to the identified risks will allow their elimination or mitigation. Conclusions: the risk management tool allowed identifying the main process-related sources of failures from the very development phase of the 100 mg succinylcoline product(AU)

Enfoque basado en riesgo desde la etapa de desarrollo del producto succinilcolina infantil 100 mg / Risk-based approach from the development phase of 100mg infant succinylcoline product

Introducción: en la actualidad las guías emitidas por la Agencia de Medicamentos y Alimentos, de los EU considera que los procesos fallan porque las fuentes no son debidamente identificadas, eliminadas o controladas y plantean un nuevo enfoque de Buenas Prácticas de Producción basado en riesgos. Objetivo: aplicar un enfoque basado en riesgos en el aseguramiento de la calidad desde la etapa de desarrollo del producto succinilcolina 100 mg. Métodos: se realizó una breve descripción del proceso mediante un diagrama de bloque, se empleó el Árbol de Fallas como herramienta para identificar las posibles fuentes de falla; la valoración de riesgos partiendo de la identificación de las posibles fuentes de fallos se ejecutó a través de un método matricial. Se identificó un orden de prioridad en la toma de acciones correctivas para eliminar o mitigar el riesgo de ocurrencia. Resultados: el diagrama de bloque permitió tener un conocimiento sobre el proceso, lo que contribuyó de forma decisiva a un mejor ejercicio de la gestión de riesgos. El Árbol de Fallas resultó útil al revelar de forma gráfica las diferentes combinaciones de fallos e interrelaciones entre causa y efecto que pudieran dar lugar al evento tope indeseado. El método empleado para la valoración de riesgos permitió determinar las prioridades, siendo el tiempo extensivo de llenado (fuera de 2-8 °C) el componente crítico a considerar con especial atención; si se tiene en cuenta que la estabilidad de este producto se puede afectar a temperaturas superiores a estas, por lo que disponer de un plan de acciones correctivas acorde a los riesgos identificados permitirá la eliminación o mitigación de las mismas. Conclusiones: la herramienta de gestión de riesgos permitió identificar desde la etapa de desarrollo del producto succinilcolina 100 mg, las principales fuentes de fallas relacionadas con este proceso(AU)

Introduction: the guidelines presently issued by the Food and Drug Agency of the United States considers that processes fail because the sources are not duly identified, eliminated or controlled and submit a new approach of Good Manufacture Practice based on risks. Objective: to apply a risk-based approach to the quality assurance from the development phase of the 100 mg succinylcoline product. Methods: the process was briefly described through a block diagram with Failure Tree as a tool for identification of possible sources of failures; the risk assessment based on the detection of the possible sources of failures was made with the matrix method. An order of priority was given in the implementation of corrective actions to eliminate or mitigate the risk of occurrence. Results: the block diagram allowed knowing the process, which contributed in a decisive way to a better application of risk management. The Failure Tree proved to be useful when showing in a graphical way the different failure combinations and interrelations between cause and effect that might give rise to the unwanted top event. The method for the risk assessment made it possible to determine priorities, being the long time of filling (not within 2 to 8oC) the critical component to be specially considered if one takes into account that stability of the product may be affected by higher temperatures, therefore, a plan of corrective actions according to the identified risks will allow their elimination or mitigation. Conclusions: the risk management tool allowed identifying the main process-related sources of failures from the very development phase of the 100 mg succinylcoline product.

Bile Acids Reduce Prion Conversion, Reduce Neuronal Loss, and Prolong Male Survival in Models of Prion Disease.

UNLABELLED: Prion diseases are fatal neurodegenerative disorders associated with the conversion of cellular prion protein (PrPC) into its aberrant infectious form (PrPSc). There is no treatment available for these diseases. The bile acids tauroursodeoxycholic acid(TUDCA) and ursodeoxycholic acid (UDCA) have been recently shown to be neuroprotective in other protein misfolding disease models, including Parkinson's, Huntington's and Alzheimer's diseases, and also in humans with amyotrophic lateral sclerosis.Here, we studied the therapeutic efficacy of these compounds in prion disease. We demonstrated that TUDCA and UDCA substantially reduced PrP conversion in cell-free aggregation assays, as well as in chronically and acutely infected cell cultures. This effect was mediated through reduction of PrPSc seeding ability, rather than an effect on PrPC. We also demonstrated the ability of TUDCA and UDCA to reduce neuronal loss in prion-infected cerebellar slice cultures. UDCA treatment reduced astrocytosis and prolonged survival in RML prion-infected mice. Interestingly, these effects were limited to the males, implying a gender-specific difference in drug metabolism. Beyond effects on PrPSc, we found that levels of phosphorylated eIF2 were increased at early time points, with correlated reductions in postsynaptic density protein 95. As demonstrated for other neurodegenerative diseases, we now show that TUDCA and UDCA may have a therapeutic role in prion diseases, with effects on both prion conversion and neuroprotection. Our findings, together with the fact that these natural compounds are orally bioavailable, permeable to the blood-brain barrier, and U.S. Food and Drug Administration-approved for use in humans, make these compounds promising alternatives for the treatment of prion diseases. IMPORTANCE: Prion diseases are fatal neurodegenerative diseases that are transmissible to humans and other mammals. There are no disease-modifying therapies available, despite decades of research. Treatment targets have included inhibition of protein accumulation,clearance of toxic aggregates, and prevention of downstream neurodegeneration. No one target may be sufficient; rather, compounds which have a multimodal mechanism, acting on different targets, would be ideal. TUDCA and UDCA are bile acids that may fulfill this dual role. Previous studies have demonstrated their neuroprotective effects in several neurodegenerative disease models, and we now demonstrate that this effect occurs in prion disease, with an added mechanistic target of upstream prion seeding. Importantly, these are natural compounds which are orally bioavailable, permeable to the blood-brain barrier, and U.S.Food and Drug Administration-approved for use in humans with primary biliary cirrhosis. They have recently been proven efficacious in human amyotrophic lateral sclerosis. Therefore, these compounds are promising options for the treatment of prion diseases.

The therapeutic potential of chemical chaperones in protein folding diseases.

Several neurodegenerative diseases are caused by defects in protein folding, including Alzheimer, Parkinson, Huntington, and prion diseases. Once a disease-specific protein misfolds, it can then form toxic aggregates which accumulate in the brain, leading to neuronal dysfunction, cell death, and clinical symptoms. Although significant advances have been made toward understanding the mechanisms of protein aggregation, there are no curative treatments for any of these diseases. Since protein misfolding and the accumulation of aggregates are the most upstream events in the pathological cascade, rescuing or stabilizing the native conformations of proteins is an obvious therapeutic strategy. In recent years, small molecules known as chaperones have been shown to be effective in reducing levels of misfolded proteins, thus minimizing the accumulation of aggregates and their downstream pathological consequences. Chaperones are classified as molecular, pharmacological, or chemical. In this mini-review we summarize the modes of action of different chemical chaperones and discuss evidence for their efficacy in the treatment of protein folding diseases in vitro and in vivo.