Advanced fluorescence imaging of in situ protein aggregation.

The aggregation of intrinsically disordered proteins is a hallmark of neurodegenerative diseases, such as Alzheimer's, Parkinson's and Huntington's disease. Although we currently have a good molecular level understanding on how protein aggregation occurs in vitro, the details of its self-assembly in live cells are still mainly unknown. During the last ten years, we have witnessed the rapid development of advanced imaging techniques, especially super-resolution and fluorescence lifetime-based microscopy, in different areas of cell biology. These methods have been revolutionising our understanding of how proteins aggregate, providing unprecedented high spatial-temporal resolution which permits us to capture the kinetics of aggregate seeding and expansion, the motion and distribution of individual aggregates within the cells, and its structural change. In this article, we will review the study of in situ protein aggregation using advanced imaging techniques, with the focus on protein aggregate structure and its assembly dynamics

From single-molecule spectroscopy to super-resolution imaging of the neuron: a review.

For more than 20 years, single-molecule spectroscopy has been providing invaluable insights into nature at the molecular level. The field has received a powerful boost with the development of the technique into super-resolution imaging methods, ca. 10 years ago, which overcome the limitations imposed by optical diffraction. Today, single molecule super-resolution imaging is routinely used in the study of macromolecular function and structure in the cell. Concomitantly, computational methods have been developed that provide information on numbers and positions of molecules at the nanometer-scale. In this overview, we outline the technical developments that have led to the emergence of localization microscopy techniques from single-molecule spectroscopy. We then provide a comprehensive review on the application of the technique in the field of neuroscience research.This work was supported by grants from the UK Engineering and Physical Sciences Research Council (EPSRC), The Wellcome Trust, Alzheimer’s Research UK, the Medical Research Council (MRC), and the Biotechnology and Biological Sciences Resesarch Council (BBSRC)

Synaptic tau: A pathological or physiological phenomenon?

In this review, we discuss the synaptic aspects of Tau pathology occurring during Alzheimer's disease (AD) and how this may relate to memory impairment, a major hallmark of AD. Whilst the clinical diagnosis of AD patients is a loss of working memory and long-term declarative memory, the histological diagnosis is the presence of neurofibrillary tangles of hyperphosphorylated Tau and Amyloid-beta plaques. Tau pathology spreads through synaptically connected neurons to impair synaptic function preceding the formation of neurofibrillary tangles, synaptic loss, axonal retraction and cell death. Alongside synaptic pathology, recent data suggest that Tau has physiological roles in the pre- or post- synaptic compartments. Thus, we have seen a shift in the research focus from Tau as a microtubule-stabilising protein in axons, to Tau as a synaptic protein with roles in accelerating spine formation, dendritic elongation, and in synaptic plasticity coordinating memory pathways. We collate here the myriad of emerging interactions and physiological roles of synaptic Tau, and discuss the current evidence that synaptic Tau contributes to pathology in AD.G.S.K.S. acknowledges funding from the Wellcome Trust (065807/Z/01/Z) (203249/Z/16/Z), the UK Medical Research Council (MRC) (MR/K02292X/1), Alzheimer Research UK (ARUK) (ARUK-PG013-14), Michael J Fox Foundation (16238) and Infnitus China Ltd. M.A.R acknowledges funding from the Engineering and Physical Sciences Research Council (EP/L015889/1)

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.We thank Prof. M. Sauer for useful discussions. This work was funded by grants from the Medical Research Council UK (MR/K015850/1 and MR/K02292X/1), Alzheimer Research UK (ARUKEG2012A-1), U.K. EPSRC (EP/H018301/1) and the Wellcome Trust (089703/Z/09/Z). D.P. wishes to acknowledge support from the Swiss National Science Foundation and the Wellcome Trust through personal fellowships. We thank LMB Visual Aids for graphics support.This is the author accepted manuscript. The final version is available from the American Chemical Society via http://dx.doi.org/10.1021/jacs.5b1101

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

Analysis of the native structure, stability and aggregation of biotinylated human lysozyme.

Fibril formation by mutational variants of human lysozyme is associated with a fatal form of hereditary non-neuropathic systemic amyloidosis. Defining the mechanistic details of lysozyme aggregation is of crucial importance for understanding the origin and progression of this disease and related misfolding conditions. In this study, we show that a biotin moiety can be introduced site-specifically at Lys33 of human lysozyme. We demonstrate, using biophysical techniques, that the structure and stability of the native-state of the protein are not detectably altered by this modification, and that the ability to form amyloid fibrils is unchanged. By taking advantage of biotin-avidin interactions, we show that super-resolution fluorescence microscopy can generate detailed images of the mature fibrils. This methodology can readily enable the introduction of additional probes into the protein, thereby providing the means through which to understand, in detail, the nature of the aggregation process of lysozyme and its variants under a variety of conditions

Modeling for policy and technology assessment: Challenges from computerbased simulations and artificial intelligence

Modeling for policy has become an integral part of policy making and technology assessment. This became particularly evident to the general public when, during the COVID-19 pandemic, forecasts of infection dynamics based on computer simulations were used to evaluate and justify policy containment measures. Computer models are also playing an increasing role in technology assessment (TA). Computer simulations are used to explore possible futures related to specific technologies, for example, in the area of energy systems analysis. Artificial intelligence (AI) models are also becoming increasingly important. The results is a mix of methods where computer simulations and machine learning converge, posing particular challenges and opening up new research questions. This Special topic brings together case studies from different fields to explore the current state of computational models in general and AI methods in particular for policy and TA