Dorothea Pinotsi

Extracellular Monomeric Tau Protein Is Sufficient to Initiate the Spread of Tau Protein Pathology

Background: The aggregation and stereotypic spreading of Tau protein is associated with Alzheimer disease. Results: Monomeric Tau enters neurons and nucleates and engages endogenous Tau to aggregate. Conclusion: Endocytosis of soluble Tau triggers aggregation in vesicles and is sufficient to initiate the spreading of pathological species. Significance: Increased levels of extracellular monomeric Tau may increase the risk of developing tauopathies. Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau.

Direct observation of heterogeneous amyloid fibril growth kinetics via two-color super-resolution microscopy.

The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases. In the present study, we show that specific events in the kinetics of the complex, multistep aggregation process of one such protein, α-synuclein, whose aggregation is a characteristic hallmark of Parkinson’s disease, can be followed at the molecular level using optical super-resolution microscopy. We have explored in particular the elongation of preformed α-synuclein fibrils; using two-color single-molecule localization microscopy we are able to provide conclusive evidence that the elongation proceeds from both ends of the fibril seeds. Furthermore, the technique reveals a large heterogeneity in the growth rates of individual fibrils; some fibrils exhibit no detectable growth, whereas others extend to more than ten times their original length within hours. These large variations in the growth kinetics can be attributed to fibril structural polymorphism. Our technique offers new capabilities in the study of amyloid growth dynamics at the molecular level and is readily translated to the study of the self-assembly of other nanostructures.

A Label‐Free, Quantitative Assay of Amyloid Fibril Growth Based on Intrinsic Fluorescence

Kinetic assay of seeded growth: The graph shows the variation in intrinsic fluorescence intensity of amyloid fibrils. Fluorescence increases during the seeded aggregation of α-synuclein seeds with α-synuclein monomeric protein (blue curve) but not when α-synuclein seeds are incubated with β-synuclein monomeric protein (black curve), thus showing that no seeded growth occurred in this case.

Proton Transfer and Structure-Specific Fluorescence in Hydrogen Bond-Rich Protein Structures.

Protein structures which form fibrils have recently been shown to absorb light at energies in the near UV range and to exhibit a structure-specific fluorescence in the visible range even in the absence of aromatic amino acids. However, the molecular origin of this phenomenon has so far remained elusive. Here, we combine ab initio molecular dynamics simulations and fluorescence spectroscopy to demonstrate that these intrinsically fluorescent protein fibrils are permissive to proton transfer across hydrogen bonds which can lower electron excitation energies and thereby decrease the likelihood of energy dissipation associated with conventional hydrogen bonds. The importance of proton transfer on the intrinsic fluorescence observed in protein fibrils is signified by large reductions in the fluorescence intensity upon either fully protonating, or deprotonating, the fibrils at pH = 0 or 14, respectively. Thus, our results point to the existence of a structure-specific fluorophore that does not require the presence of aromatic residues or multiple bond conjugation that characterize conventional fluorescent systems. The phenomenon may have a wide range of implications in biological systems and in the design of self-assembled functional materials.

Highly potent soluble amyloid-β seeds in human Alzheimer brain but not cerebrospinal fluid

The soluble fraction of brain samples from patients with Alzheimers disease contains highly biologically active amyloid-β seeds. In this study, we sought to assess the potency of soluble amyloid-β seeds derived from the brain and cerebrospinal fluid. Soluble Alzheimers disease brain extracts were serially diluted and then injected into the hippocampus of young, APP transgenic mice. Eight months later, seeded amyloid-β deposition was evident even when the hippocampus received subattomole amounts of brain-derived amyloid-β. In contrast, cerebrospinal fluid from patients with Alzheimers disease, which contained more than 10-fold higher levels of amyloid-β peptide than the most concentrated soluble brain extracts, did not induce detectable seeding activity in vivo. Similarly, cerebrospinal fluid from aged APP-transgenic donor mice failed to induce cerebral amyloid-β deposition. In comparison to the soluble brain fraction, cerebrospinal fluid largely lacked N-terminally truncated amyloid-β species and exhibited smaller amyloid-β-positive particles, features that may contribute to the lack of in vivo seeding by cerebrospinal fluid. Interestingly, the same cerebrospinal fluid showed at least some seeding activity in an in vitro assay. The present results indicate that the biological seeding activity of soluble amyloid-β species is orders of magnitude greater in brain extracts than in the cerebrospinal fluid.

Blind assessment of localisation microscope image resolution

BackgroundThis paper analyses the resolution achieved in localisation microscopy experiments. The resolution is an essential metric for the correct interpretation of super-resolution images, but it varies between specimens due to different localisation precisions and densities.MethodsBy analysing localisation microscopy as a statistical method of Density Estimation, we present a method that produces a blind estimate of the resolution in a super-resolved image. This estimate is derived directly from the raw image data without the need for comparisons with known calibration specimens. It is corroborated with simulated and experimental data.Results and discussionLocalisation microscopy has a resolution limit equal to 2σ, where σ is the r.m.s. localisation precision, evaluated as an average Thompson precision, Cramer Rao bound, or otherwise. Further, for a limited-sampling case in which there is only one localisation per fluorophore, the expected resolution of an optimised super-resolution image is worsened to approximately 3σ, due to smoothing processes that are necessarily involved in visualising the specimen with limited data. This 2σ or 3σ resolution can be estimated for any localisation microscopy specimen, and this metric can corroborate or replace empirical estimates of resolution. Other quantifiable resolution losses arise from sparse labelling, fluorescent label size, and motion blur.

Single Photon Absorption by a Single Quantum Emitter

We show that a three-level lambda quantum emitter with equal spontaneous emission rates on both optically active transitions can absorb an incident single-photon pulse with a probability approaching unity, provided that the focused light profile matches that of the emitter dipole emission. Even with realistic focusing geometries, our results could find applications in long-distance entanglement of spin qubits.

Nanoscopic insights into seeding mechanisms and toxicity of α-synuclein species in neurons

Significance The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders. Here, we monitor the fate of different forms of α-synuclein (AS), a protein implicated in Parkinson’s disease, via optical nanoscopy directly in neuronal cells. We show that exogenously added preformed AS fibrils elongate by the addition of endogenous AS, naturally present in neurons. In contrast, exogenously added monomeric AS induces aggregate formation within the cells and leads to apoptosis. The latter is significantly reduced by the addition of preformed fibrils, suggesting a neuroprotective role of fibrillar species. The visualization of these effects at the nanoscale shown here opens up new avenues for understanding the links between AS aggregation and neuronal toxicity. New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinsons and Alzheimers diseases. Here, we probe the links between the mechanism of α-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson’s disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.

Optical Super-Resolution Imaging of β-Amyloid Aggregation In Vitro and In Vivo: Method and Techniques

Super-resolution microscopy has emerged as a powerful and non-invasive tool for the study of molecular processes both in vitro and in live cells. In particular, super-resolution microscopy has proven valuable for research studies in protein aggregation. In this chapter we present details of recent advances in this method and the specific techniques, enabling the study of amyloid beta aggregation optically, both in vitro and in cells. First, we show that variants of optical super-resolution microscopy provide a capability to visualize oligomeric and fibrillar structures directly, providing detailed information on species morphology in vitro and even in situ, in the cellular environment. We focus on direct Stochastic Optical Reconstruction Microscopy, dSTORM, which provides morphological detail on spatial scales below 20 nm, and provide detailed protocols for its implementation in the context of amyloid beta research. Secondly, we present a range of optical techniques that offer super-resolution indirectly, which we call multi-parametric microscopy. The latter offers molecular scale information on self-assembly reactions via changes in protein or fluorophore spectral signatures. These techniques are empowered by our recent discovery that disease related amyloid proteins adopt intrinsic energy states upon fibrilisation. We show that fluorescence lifetime imaging provides a particularly sensitive readout to report on the aggregation state, which is robustly quantifiable for experiments performed either in vitro or in vivo.

Resonant Spectroscopy on Charge Tunable Quantum Dots in Photonic Crystal Structures

We demonstrate resonant excitation of a semiconductor quantum dot embedded in a p-i-n diode structure in and out of a photonic crystal cavity. We investigate the modification of the spontaneous emission rate and the photon collection efficiency of quantum dots due to the presence of a photonic bandgap. The resonant excitation together with deterministic charging of the quantum dot embedded in a photonic crystal, can be used for single electron spin measurement.