Claire H. Michel

ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function

Summary The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

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.

Characterisation of serpin polymers in vitro and in vivo

Neuroserpin is a member of the serine protease inhibitor or serpin superfamily of proteins. It is secreted by neurones and plays an important role in the regulation of tissue plasminogen activator at the synapse. Point mutations in the neuroserpin gene cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. This is one of a group of disorders caused by mutations in the serpins that are collectively known as the serpinopathies. Others include α(1)-antitrypsin deficiency and deficiency of C1 inhibitor, antithrombin and α(1)-antichymotrypsin. The serpinopathies are characterised by delays in protein folding and the retention of ordered polymers of the mutant serpin within the cell of synthesis. The clinical phenotype results from either a toxic gain of function from the inclusions or a loss of function, as there is insufficient protease inhibitor to regulate important proteolytic cascades. We describe here the methods required to characterise the polymerisation of neuroserpin and draw parallels with the polymerisation of α(1)-antitrypsin. It is important to recognise that the conditions in which experiments are performed will have a major effect on the findings. For example, incubation of monomeric serpins with guanidine or urea will produce polymers that are not found in vivo. The characterisation of the pathological polymers requires heating of the folded protein or alternatively the assessment of ordered polymers from cell and animal models of disease or from the tissues of humans who carry the mutation.

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.

C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction

Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.Alpha-synuclein is associated with neuronal dysfunction in Parkinson’s disease. This study shows that alpha-synuclein interacts with neuronal synaptic vesicles in a calcium-dependent fashion, and this interaction is important for synaptic vesicle clustering.

Advanced imaging of tau pathology in Alzheimer Disease: New perspectives from super resolution microscopy and label‐free nanoscopy

Alzheimers disease (AD) is the main cause of dementia in the elderly population. Over 30 million people worldwide are living with dementia and AD prevalence is projected to increase dramatically in the next two decades. In terms of neuropathology, AD is characterized by two major cerebral hallmarks: extracellular β‐amyloid (Aβ) plaques and intracellular Tau inclusions, which start accumulating in the brain 15‐20 years before the onset of symptoms. Within this context, the scientific community worldwide is undertaking a wide research effort to detect AD pathology at its earliest, before symptoms appear. Neuroimaging of Aβ by positron emission tomography (PET) is clinically available and is a promising modality for early detection of Aβ pathology and AD diagnosis. Substantive efforts are ongoing to develop advanced imaging techniques for early detection of Tau pathology. Here, we will briefly describe the key features of Tau pathology and its heterogeneity across various neurodegenerative diseases bearing cerebral Tau inclusions (i.e., tauopathies). We will outline the current status of research on Tau‐specific PET tracers and their clinical development. Finally, we will discuss the potential application of novel super‐resolution and label‐free techniques for investigating Tau pathology at the experimental level and their potential application for AD diagnosis. Microsc. Res. Tech. 79:677–683, 2016.

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in C. elegans

Reduced protein homeostasis and increased protein instability is a common feature of aging. Yet it remains unclear whether protein instability is a cause of aging. In neurodegenerative diseases and amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological solid aggregates in a variety of tissues. More recently, widespread protein aggregation has been described during normal aging, in the absence of disease processes. Until now, an extensive characterization of the nature of age-dependent protein aggregation and its consequences for aging has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and contain amyloid-like structures similar to disease-associated protein aggregates. Moreover, we demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in C. elegans. Together, these finding reveal that the formation of amyloid aggregates is a generic problem of aging and likely to be an important target for strategies designed to maintain physiological functions in later stages of life.

Calcium binding at the C-terminus of α-synuclein modulates synaptic vesicle interaction

J.L. was supported by a research fellowship from the Deutsche Forschungsgemeinschaft (DFG; award LA 3609/2-1). M.Z. acknowledges funding from the Eugenides Foundation. C.F.K. acknowledges funding from the UK Engineering and Physical Sciences Research Council (EPSRC). A.DS. acknowledges funding from the UK Medical Research Council (MRC, MR/N000676/1). A.DS. and G.F. acknowledge funding from Parkinsons UK (G-1508). G.S.K. and C.F.K. acknowledge funding from the Wellcome Trust, the UK Medical Research Council (MRC), Alzheimer Research UK (ARUK), and Infinitus China Ltd. J.L. and A.D.S. acknowledge Alzheimer Research UK (ARUK) travel grants.

Nanoscale imaging of neurotoxic proteins

The misfolding and self-assembly of intrinsically disordered proteins into insoluble amyloid structures is central to many neurodegenerative diseases such as Alzheimer’s and Parkinson’s Diseases. Optical imaging of this self-assembly process in vitro and in cells is revolutionising our understanding of the molecular mechanisms behind these devastating diseases. In contrast to conventional biophysical methods, optical imaging, and in particular optical super-resolution imaging, permit the dynamic investigation of the molecular self-assembly process in vitro and in cells, at molecular level resolution. In this article, current state-of-the-art imaging methods are reviewed and discussed in the context of research into neurodegeneration.

In situ studies of protein aggregation kinetics with multiparametric and superresolution imaging

Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Parkinson’s (PD) and Alzheimer’s disease (AD). Their common feature is that normally unstructured and soluble proteins misfold and aggregate into insoluble amyloid fibrils, which make up the deposits in the brains of patients suffering from these devastating illnesses. A key requirement to gain insight into molecular mechanisms of disease and to progress in the search for therapeutic intervention is a capability to image the aggregation process and structure of ensuing fibrils in situ. We have recently reported that amyloid proteins that are associated with protein misfolding diseases, including PD, AD and various other types of amyloidosis develop an intrinsic fluorescence in the visible range [1,2]. The discovery of intrinsic amyloid fluorescence has enabled the process of amyloid formation from disease-relevant polypeptides to be monitored in a label-free manner and with high specificity [2,3]. I will show here how specific and sensitive in vivo probes of amyloid structures can be developed using external fluorophores covalently attached to the amyloid backbone. Such external fluorophores can participate in Forster resonance energy transfer (FRET) with intrinsic energy states of amyloid structures if present, providing a readout in the form of a reduced fluorescence lifetime of the external fluorophores. I will provide an overview on the application of all-optical techniques to inform on the aggregation state of α-synuclein (relevant to PD), amyloid β and Tau (relevant to AD) in vitro, in live cells and model organisms. Multiparametric microscopy permits us to follow the kinetics of amyloid oligomerisation in vivo and correlate the appearance of aggregates with phenotypes of disease [1]. Using direct stochastic optical reconstruction microscopy, dSTORM, we are able to probe, in cells, the morphology of the ensuing aggregates with a resolution better than 20 nm [4]. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease.