Wild-type sTREM2 blocks Aß aggregation and neurotoxicity, but the Alzheimer's R47H mutant increases Aß aggregation.

TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aß and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aß oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aß oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aß oligomerization and disaggregated preformed Aß oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aß fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aß-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal-glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aß but rather promotes Aß protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aß-induced release of sTREM2, which blocks Aß aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aß aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk.

Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity.

Observation of highly dynamic processes inside living cells at the single molecule level is key for a better understanding of biological systems. However, imaging of single molecules in living cells is usually limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination, for example, in a reflected geometry with a high numerical aperture imaging objective, have been developed. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-colour acquisition of z -stacks. We demonstrate fast volume scanning by imaging a two-colour giant unilamellar vesicle (GUV) hemisphere. In addition, the high contrast enabled the imaging and tracking of single lipids in the GUV cap. The enhanced reflected scanning light-sheet microscope enables fast 3D scanning of artificial membrane systems and potentially live cells with single-molecule sensitivity and thereby could provide quantitative and molecular insight into the operation of cells.

Safety and immunogenicity of a tetravalent dengue vaccine in children aged 2-17 years: a randomised, placebo-controlled, phase 2 trial.

BACKGROUND: An unmet clinical need remains for an effective tetravalent dengue vaccine suitable for all age groups, regardless of serostatus. We assessed the immunogenicity and safety of three different dose schedules of a tetravalent dengue vaccine (TAK-003) over a 48-month period in children living in dengue-endemic countries. METHODS: We did a large, phase 2, double-blind, placebo-controlled trial at three sites in the Dominican Republic, Panama, and the Philippines. Healthy participants aged 2-17 years were randomly assigned 1:2:5:1 using an interactive web response system with stratification by age to receive either a two-dose primary series (days 1 and 91), one primary dose (day 1), one primary dose plus booster (days 1 and 365), or placebo. Participants and relevant study personnel were masked to the random assignment until completion of the study at month 48. To maintain masking, TAK-003 recipients were administered placebo doses when appropriate. The primary objective was assessment of neutralising geometric mean titres for each serotype to month 48 assessed in the per-protocol immunogenicity subset. Secondary safety endpoints included proportions of participants with serious adverse events and symptomatic virologically confirmed dengue. This study is registered with ClinicalTrials.gov, NCT02302066. FINDINGS: Between Dec 5, 2014, and Feb 13, 2015, 1800 children were randomly assigned to the following groups: two-dose primary series (n=201), one primary dose (n=398), one primary dose plus 1-year booster (n=1002), and placebo (n=199). Of them, 1479 (82%) participants completed the 48-month study. Immunogenicity endpoints were assessed in 562 participants enrolled in the immunogenicity subset, of whom 509 were included in the per-protocol subset. At month 48, antibody titres remained elevated in all TAK-003 groups compared with placebo, irrespective of baseline serostatus. At month 48, geometric mean titres were 378 (95% CI 226-632) in two-dose, 421 (285-622) in one-dose, 719 (538-960) in one-dose plus 1-year booster, and 100 (50-201) in placebo recipients against DENV 1; 1052 (732-1511), 1319 (970-1794), 1200 (927-1553), and 208 (99-437) against DENV 2; 183 (113-298), 201 (135-298), 288 (211-392), and 71 (37-139) against DENV 3; and 152 (97-239), 164 (114-236), 219 (165-290), and 46 (26-82) against DENV 4; and tetravalent seropositivity rate was 89% (79-96), 86% (80-92), 97% (93-99), and 60% (47-72), respectively. Virologically confirmed dengue was recorded in 37 (2%) TAK-003 and 13 (7%) placebo participants, with a relative risk of 0·35 (0·19-0·65). No vaccine-related serious adverse events or severe dengue virus disease were reported. INTERPRETATION: TAK-003 elicited antibody responses against all four serotypes, which persisted to 48 months post-vaccination, regardless of baseline serostatus. No important safety risks were identified. We observed a long-term reduction in risk of symptomatic dengue virus disease in vaccinees. Results from this study provide a long-term safety database and support assessment of the vaccine in the ongoing phase 3 efficacy study. FUNDING: Takeda Vaccines.

Mapping Surface Hydrophobicity of α-Synuclein Oligomers at the Nanoscale.

Proteins fold into a single structural ensemble but can also misfold into many diverse structures including small aggregates and fibrils, which differ in their toxicity. The aggregate surface properties play an important role in how they interact with the plasma membrane and cellular organelles, potentially inducing cellular toxicity, however, these properties have not been measured to date due to the lack of suitable methods. Here, we used a spectrally resolved, super-resolution imaging method combined with an environmentally sensitive fluorescent dye to measure the surface hydrophobicity of individual aggregates formed by the protein α-synuclein (αS), whose aggregation is associated with Parkinson's disease. We show that the surface of soluble oligomers is more hydrophobic than fibrils and populates a diverse range of coexisting states. Overall, our data show that the conversion of oligomers to fibril-like aggregates and ultimately to fibrils results in a reduction in both hydrophobicity and the variation in hydrophobicity. This funneling characteristic of the energy landscape explains many of the observed properties of αS aggregates and may be a common feature of aggregating proteins.

Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells.

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid-specific aptamer, we demonstrate that this technique is able to detect and super-resolve a range of aggregated species, including those formed by α-synuclein and amyloid-ß. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.

Optical Structural Analysis of Individual α-Synuclein Oligomers.

Small aggregates of misfolded proteins play a key role in neurodegenerative disorders. Such species have proved difficult to study due to the lack of suitable methods capable of resolving these heterogeneous aggregates, which are smaller than the optical diffraction limit. We demonstrate here an all-optical fluorescence microscopy method to characterise the structure of individual protein aggregates based on the fluorescence anisotropy of dyes such as thioflavin-T, and show that this technology is capable of studying oligomers in human biofluids such as cerebrospinal fluid. We first investigated in vitro the structural changes in individual oligomers formed during the aggregation of recombinant α-synuclein. By studying the diffraction-limited aggregates we directly evaluated their structural conversion and correlated this with the potential of aggregates to disrupt lipid bilayers. We finally characterised the structural features of aggregates present in cerebrospinal fluid of Parkinson's disease patients and age-matched healthy controls.

Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates.

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aß42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aß peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.

Co-aggregation with Apolipoprotein E modulates the function of Amyloid-ß in Alzheimer's disease.

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-ß (Aß) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aß in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aß co-aggregates account for ~50% of the mass of diffusible Aß aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aß tune disease-related functions of Aß aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aß. Selectively removing non-lipidated apoE4-Aß co-aggregates enhances clearance of toxic Aß by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

Ultranarrow and widely tunable Mn2+-Induced photoluminescence from single Mn-doped nanocrystals of ZnS-CdS alloys.

Extensively studied Mn-doped semiconductor nanocrystals have invariably exhibited photoluminescence over a narrow energy window of width ≤150 meV in the orange-red region and a surprisingly large spectral width (≥180 meV), contrary to its presumed atomic-like origin. Carrying out emission measurements on individual single nanocrystals and supported by ab initio calculations, we show that Mn PL emission, in fact, can (i) vary over a much wider range (∼370 meV) covering the deep green--deep red region and (ii) exhibit widths substantially lower (∼60-75 meV) than reported so far, opening newer application possibilities and requiring a fundamental shift in our perception of the emission from Mn-doped semiconductor nanocrystals.

Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent responses.

Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3ß or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies.

Small soluble α-synuclein aggregates are the toxic species in Parkinson's disease.

Soluble α-synuclein aggregates varying in size, structure, and morphology have been closely linked to neuronal death in Parkinson's disease. However, the heterogeneity of different co-existing aggregate species makes it hard to isolate and study their individual toxic properties. Here, we show a reliable non-perturbative method to separate a heterogeneous mixture of protein aggregates by size. We find that aggregates of wild-type α-synuclein smaller than 200 nm in length, formed during an in vitro aggregation reaction, cause inflammation and permeabilization of single-liposome membranes and that larger aggregates are less toxic. Studying soluble aggregates extracted from post-mortem human brains also reveals that these aggregates are similar in size and structure to the smaller aggregates formed in aggregation reactions in the test tube. Furthermore, we find that the soluble aggregates present in Parkinson's disease brains are smaller, largely less than 100 nm, and more inflammatory compared to the larger aggregates present in control brains. This study suggests that the small non-fibrillar α-synuclein aggregates are the critical species driving neuroinflammation and disease progression.

Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity.

Aggregation of alpha-synuclein (α-Syn) drives Parkinson's disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)-derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

Soluble amyloid beta-containing aggregates are present throughout the brain at early stages of Alzheimer's disease.

Protein aggregation likely plays a key role in the initiation and spreading of Alzheimer's disease pathology through the brain. Soluble aggregates of amyloid beta are believed to play a key role in this process. However, the aggregates present in humans are still poorly characterized due to a lack of suitable methods required for characterizing the low concentration of heterogeneous aggregates present. We have used a variety of biophysical methods to characterize the aggregates present in human Alzheimer's disease brains at Braak stage III. We find soluble amyloid beta-containing aggregates in all regions of the brain up to 200 nm in length, capable of causing an inflammatory response. Rather than aggregates spreading through the brain as disease progresses, it appears that aggregation occurs all over the brain and that different brain regions are at earlier or later stages of the same process, with the later stages causing increased inflammation.

Tumour necrosis factor induces increased production of extracellular amyloid-ß- and α-synuclein-containing aggregates by human Alzheimer's disease neurons.

In addition to increased aberrant protein aggregation, inflammation has been proposed as a key element in the pathogenesis and progression of Alzheimer's disease. How inflammation interacts with other disease pathways and how protein aggregation increases during disease are not clear. We used single-molecule imaging approaches and membrane permeabilization assays to determine the effect of chronic exposure to tumour necrosis factor, a master proinflammatory cytokine, on protein aggregation in human-induced pluripotent stem cell-derived neurons harbouring monogenic Alzheimer's disease mutations. We report that exposure of Alzheimer's disease neurons, but not control neurons, to tumour necrosis factor induces substantial production of extracellular protein aggregates. Aggregates from Alzheimer's disease neurons are composed of amyloid-ß and α-synuclein and induce significant permeabilization of lipid membranes in an assay of pathogenicity. These findings provide support for a causal relationship between two crucial processes in Alzheimer's disease pathogenesis and suggest that targeting inflammation, particularly tumour necrosis factor, may have beneficial downstream effects on ameliorating aberrant protein aggregation and accumulation.

Direct measurement of lipid membrane disruption connects kinetics and toxicity of Aß42 aggregation.

The formation of amyloid deposits in human tissues is a defining feature of more than 50 medical disorders, including Alzheimer's disease. Strong genetic and histological evidence links these conditions to the process of protein aggregation, yet it has remained challenging to identify a definitive connection between aggregation and pathogenicity. Using time-resolved fluorescence microscopy of individual synthetic vesicles, we show for the Aß42 peptide implicated in Alzheimer's disease that the disruption of lipid bilayers correlates linearly with the time course of the levels of transient oligomers generated through secondary nucleation. These findings indicate a specific role of oligomers generated through the catalytic action of fibrillar species during the protein aggregation process in driving deleterious biological function and establish a direct causative connection between amyloid formation and its pathological effects.

Analysis of α-synuclein species enriched from cerebral cortex of humans with sporadic dementia with Lewy bodies.

Since researchers identified α-synuclein as the principal component of Lewy bodies and Lewy neurites, studies have suggested that it plays a causative role in the pathogenesis of dementia with Lewy bodies and other 'synucleinopathies'. While α-synuclein dyshomeostasis likely contributes to the neurodegeneration associated with the synucleinopathies, few direct biochemical analyses of α-synuclein from diseased human brain tissue currently exist. In this study, we analysed sequential protein extracts from a substantial number of patients with neuropathological diagnoses of dementia with Lewy bodies and corresponding controls, detecting a shift of cytosolic and membrane-bound physiological α-synuclein to highly aggregated forms. We then fractionated aqueous extracts (cytosol) from cerebral cortex using non-denaturing methods to search for soluble, disease-associated high molecular weight species potentially associated with toxicity. We applied these fractions and corresponding insoluble fractions containing Lewy-type aggregates to several reporter assays to determine their bioactivity and cytotoxicity. Ultimately, high molecular weight cytosolic fractions enhances phospholipid membrane permeability, while insoluble, Lewy-associated fractions induced morphological changes in the neurites of human stem cell-derived neurons. While the concentrations of soluble, high molecular weight α-synuclein were only slightly elevated in brains of dementia with Lewy bodies patients compared to healthy, age-matched controls, these observations suggest that a small subset of soluble α-synuclein aggregates in the brain may drive early pathogenic effects, while Lewy body-associated α-synuclein can drive neurotoxicity.

Alpha synuclein aggregation drives ferroptosis: an interplay of iron, calcium and lipid peroxidation.

Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of ß-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson's disease, and highlights a new mechanism by which lipid peroxidation causes cell death.

Correction: Alpha synuclein aggregation drives ferroptosis: an interplay of iron, calcium and lipid peroxidation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Imaging individual protein aggregates to follow aggregation and determine the role of aggregates in neurodegenerative disease.

Protein aggregates play a key role in the initiation and spreading of neurodegenerative disease but have been difficult to study due to their low abundance and heterogeneity, in both size and structure. Fluorescence based methods capable of detecting and characterising single aggregates have recently been developed and can be used to measure many important aggregate properties, and can be combined with sensitive assays to measure aggregate toxicity. Here we review these methods and discuss recent examples of their application to determine the molecular mechanism of aggregation and the detection of aggregates in cells and cerebrospinal fluid. The further development of these methods and their application to the aggregates present in humans has the potential to solve a major problem in the field and allow the identification of the key toxic species that should be targeted in therapies.

Direct observation of prion protein oligomer formation reveals an aggregation mechanism with multiple conformationally distinct species.

The aggregation of the prion protein (PrP) plays a key role in the development of prion diseases. In the past decade, a similar process has been associated with other proteins, such as Aß, tau, and α-synuclein, which participate in other neurodegenerative diseases. It is increasingly recognized that the small oligomeric species of aggregates can play an important role in the development of prion diseases. However, determining the nature of the oligomers formed during the aggregation process has been experimentally difficult due to the lack of suitable methods capable of the detection and characterization of the low level of oligomers that may form. To address this problem, we have utilized single-aggregate methods to study the early events associated with aggregation of recombinant murine PrP in vitro to approach the bona fide process in vivo. PrP aggregation resulted in the formation of thioflavin T (ThT)-inactive and ThT-active species of oligomers. The ThT-active oligomers undergo conversion from a Proteinase K (PK)-sensitive to PK-resistant conformer, from which mature fibrils can eventually emerge. Overall, our results show that single-aggregate methods can provide structural and mechanistic insights into PrP aggregation, identify the potential species that mediates cytotoxicity, and reveal that a range of distinct oligomeric species with different properties is formed during prion protein aggregation.