Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational ...change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.
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► A variety of oligomers form during αS amyloid fibril assembly and disaggregation ► A slow process converts early oligomers to stable, proteinase-K-resistant forms ► Oligomers arising from the conversion reaction are more toxic than the early species ► Mature amyloid fibrils can either sequester or release potentially cytotoxic oligomers
Single-molecule fluorescence reveals that the cytotoxic species of α-synuclein associated with Parkinson's disease forms via the slow structural conversion of disordered, nontoxic oligomers into a stable form that is toxic to primary neurons and that precedes fibril formation.
Roboticists have begun to design biologically inspired robots with soft or partially soft bodies, which have the potential to be more robust and adaptable, and safer for human interaction, than ...traditional rigid robots. However, key challenges in the design and manufacture of soft robots include the complex fabrication processes and the interfacing of soft and rigid components. We used multimaterial three-dimensional (3D) printing to manufacture a combustion-powered robot whose body transitions from a rigid core to a soft exterior. This stiffness gradient, spanning three orders of magnitude in modulus, enables reliable interfacing between rigid driving components (controller, battery, etc.) and the primarily soft body, and also enhances performance. Powered by the combustion of butane and oxygen, this robot is able to perform untethered jumping.
Abstract
Advances in the technologies and informatics used to generate and process large biological data sets (omics data) are promoting a critical shift in the study of biomedical sciences. While ...genomics, transcriptomics and proteinomics, coupled with bioinformatics and biostatistics, are gaining momentum, they are still, for the most part, assessed individually with distinct approaches generating monothematic rather than integrated knowledge. As other areas of biomedical sciences, including metabolomics, epigenomics and pharmacogenomics, are moving towards the omics scale, we are witnessing the rise of inter-disciplinary data integration strategies to support a better understanding of biological systems and eventually the development of successful precision medicine. This review cuts across the boundaries between genomics, transcriptomics and proteomics, summarizing how omics data are generated, analysed and shared, and provides an overview of the current strengths and weaknesses of this global approach. This work intends to target students and researchers seeking knowledge outside of their field of expertise and fosters a leap from the reductionist to the global-integrative analytical approach in research.
Cells respond to endolysosome damage by either repairing the damage or targeting damaged endolysosomes for degradation via lysophagy. However, the signals regulating the decision for repair or ...lysophagy are poorly characterised. Here, we show that the Parkinson's disease (PD)‐related kinase LRRK2 is activated in macrophages by pathogen‐ or sterile‐induced endomembrane damage. LRRK2 recruits the Rab GTPase Rab8A to damaged endolysosomes as well as the ESCRT‐III component CHMP4B, thereby favouring ESCRT‐mediated repair. Conversely, in the absence of LRRK2 and Rab8A, damaged endolysosomes are targeted to lysophagy. These observations are recapitulated in macrophages from PD patients where pathogenic LRRK2 gain‐of‐function mutations result in the accumulation of endolysosomes which are positive for the membrane damage marker Galectin‐3. Altogether, this work indicates that LRRK2 regulates endolysosomal homeostasis by controlling the balance between membrane repair and organelle replacement, uncovering an unexpected function for LRRK2, and providing a new link between membrane damage and PD.
Synopsis
Leucine‐rich repeat kinase 2 (LRRK2) has been linked to neurodegenerative and inflammatory diseases but it is unknown how LRKK2 activation occurs in these pathological conditions. Here, pathogen‐ or drug‐induced endomembrane damage is found to activate LRRK2 in macrophages, which in turn controls recruitment of membrane repair factors.
LRRK2 is activated upon endomembrane damage.
Endomembrane damage triggers LRRK2‐mediated phosphorylation of Rab8A.
Rab8A and ESCRT component CHMP4B are recruited to damaged endolysosomes in a LRRK2‐dependent manner.
In the absence of LRRK2 and Rab8A, damaged endolysosomes are disposed through lysophagy.
Macrophages from patients harbouring LRRK2 G2019S or R1441C mutation show accumulation of damaged endolysosomes.
LRRK2 phosphorylation of Rab8A GTPase promotes its co‐translocation with ESCRT component CHMP4B to damaged endolysosomes for their repair.
In this study, we combined linkage analysis with whole-exome sequencing of two individuals to identify candidate causal variants in a moderately-sized UK kindred exhibiting autosomal-dominant ...inheritance of craniocervical dystonia. Subsequent screening of these candidate causal variants in a large number of familial and sporadic cases of cervical dystonia led to the identification of a total of six putatively pathogenic mutations in ANO3, a gene encoding a predicted Ca2+-gated chloride channel that we show to be highly expressed in the striatum. Functional studies using Ca2+ imaging in case and control fibroblasts demonstrated clear abnormalities in endoplasmic-reticulum-dependent Ca2+ signaling. We conclude that mutations in ANO3 are a cause of autosomal-dominant craniocervical dystonia. The locus DYT23 has been reserved as a synonym for this gene. The implication of an ion channel in the pathogenesis of dystonia provides insights into an alternative mechanism that opens fresh avenues for further research.
Devices fabricated using soft materials have been a major research focus of late, capturing the attention of scientists and laypersons alike in a wide range of fields, from microfluidics to robotics. ...The functionality of such devices relies on their structural and material properties; thus, the fabrication method is of utmost importance. Here, multilayer soft lithography, precision laser micromachining, and folding to establish a new paradigm are combined for creating 3D soft microstructures and devices. Phase‐changing materials are exploited to transform actuators into structural elements, allowing 2D laminates to evolve into a third spatial dimension. To illustrate the capabilities of this new fabrication paradigm, the first “microfluidic origami for reconfigurable pneumatic/hydraulic” device is designed and manufactured: a 12‐layer soft robotic peacock spider with embedded microfluidic circuitry and actuatable features.
Multilayer soft lithography, precision laser micromachining, and folding are combined to establish a new paradigm for creating 3D soft microstructures and devices. Phase‐changing materials are exploited to transform actuators into structural elements, allowing 2D laminates to evolve into a third spatial dimension. This results in a 12‐layer soft robotic peacock spider with embedded microfluidic circuitry and actuatable features.
Exome sequencing has proven to be an effective tool to discover the genetic basis of Mendelian disorders. It is well established that copy number variants (CNVs) contribute to the etiology of these ...disorders. However, calling CNVs from exome sequence data is challenging. A typical read depth strategy consists of using another sample (or a combination of samples) as a reference to control for the variability at the capture and sequencing steps. However, technical variability between samples complicates the analysis and can create spurious CNV calls.
Here, we introduce ExomeDepth, a new CNV calling algorithm designed to control for this technical variability. ExomeDepth uses a robust model for the read count data and uses this model to build an optimized reference set in order to maximize the power to detect CNVs. As a result, ExomeDepth is effective across a wider range of exome datasets than the previously existing tools, even for small (e.g. one to two exons) and heterozygous deletions. We used this new approach to analyse exome data from 24 patients with primary immunodeficiencies. Depending on data quality and the exact target region, we find between 170 and 250 exonic CNV calls per sample. Our analysis identified two novel causative deletions in the genes GATA2 and DOCK8.
The code used in this analysis has been implemented into an R package called ExomeDepth and is available at the Comprehensive R Archive Network (CRAN).
Significance Certain oligomeric species generated during the self-assembly of specific proteins into ordered fibrillar aggregates are likely to be key players in the initiation and spreading of ...neurodegenerative diseases. We have purified stable toxic oligomeric species of α-synuclein and defined and minimized their degree of heterogeneity, which has allowed us to identify distinct subgroups of oligomers and determine their structural properties and three-dimensional molecular architectures. All the oligomeric subgroups possess approximately cylindrical architectures with marked similarities to amyloid fibrils, suggesting that these types of oligomers are kinetically trapped during protein self-assembly. The relative stabilities and inherent pathological roles of different amyloid oligomers are likely to result from the multiplicity of pathways of the misfolding process and the remarkably slow rates of structural conversions.
We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.
Both positive and negative associations between higher body mass index (BMI) and Parkinson disease (PD) have been reported in observational studies, but it has been difficult to establish causality ...because of the possibility of residual confounding or reverse causation. To our knowledge, Mendelian randomisation (MR)-the use of genetic instrumental variables (IVs) to explore causal effects-has not previously been used to test the effect of BMI on PD.
Two-sample MR was undertaken using genome-wide association (GWA) study data. The associations between the genetic instruments and BMI were obtained from the GIANT consortium and consisted of the per-allele difference in mean BMI for 77 independent variants that reached genome-wide significance. The per-allele difference in log-odds of PD for each of these variants was estimated from a recent meta-analysis, which included 13,708 cases of PD and 95,282 controls. The inverse-variance weighted method was used to estimate a pooled odds ratio (OR) for the effect of a 5-kg/m2 higher BMI on PD. Evidence of directional pleiotropy averaged across all variants was sought using MR-Egger regression. Frailty simulations were used to assess whether causal associations were affected by mortality selection. A combined genetic IV expected to confer a lifetime exposure of 5-kg/m2 higher BMI was associated with a lower risk of PD (OR 0.82, 95% CI 0.69-0.98). MR-Egger regression gave similar results, suggesting that directional pleiotropy was unlikely to be biasing the result (intercept 0.002; p = 0.654). However, the apparent protective influence of higher BMI could be at least partially induced by survival bias in the PD GWA study, as demonstrated by frailty simulations. Other important limitations of this application of MR include the inability to analyse non-linear associations, to undertake subgroup analyses, and to gain mechanistic insights.
In this large study using two-sample MR, we found that variants known to influence BMI had effects on PD in a manner consistent with higher BMI leading to lower risk of PD. The mechanism underlying this apparent protective effect warrants further study.
Reports of primary isolated dystonia inherited in an autosomal-recessive (AR) manner, often lumped together as “DYT2 dystonia,” have appeared in the scientific literature for several decades, but no ...genetic cause has been identified to date. Using a combination of homozygosity mapping and whole-exome sequencing in a consanguineous kindred affected by AR isolated dystonia, we identified homozygous mutations in HPCA, a gene encoding a neuronal calcium sensor protein found almost exclusively in the brain and at particularly high levels in the striatum, as the cause of disease in this family. Subsequently, compound-heterozygous mutations in HPCA were also identified in a second independent kindred affected by AR isolated dystonia. Functional studies suggest that hippocalcin might play a role in regulating voltage-dependent calcium channels. The identification of mutations in HPCA as a cause of AR primary isolated dystonia paves the way for further studies to assess whether “DYT2 dystonia” is a genetically homogeneous condition or not.
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