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The 42-amino-acid β-amyloid (Aβ42) is a critical causative agent in the pathology of Alzheimer's disease. The hereditary Arctic mutation of Aβ42 (E22G) leads to increased intracellular accumulation of β-amyloid in early-onset Alzheimer's disease. However, it remains largely unknown how the Arctic mutant variant leads to aggressive protein aggregation and increased intracellular toxicity. Here, we constructed stable cell lines expressing fluorescent-tagged wildtype (WT) and E22G Aβ42 to study the aggregation kinetics of the Arctic Aβ42 mutant peptide and its heterogeneous structural forms. Arctic-mutant peptides assemble and form fibrils at a much faster rate than WT peptides. We identified five categories of intracellular aggregate-oligomers, single fibrils, fibril bundles, clusters, and aggresomes-that underline the heterogeneity of these Aβ42 aggregates and represent the progression of Aβ42 aggregation within the cell. Fluorescence-lifetime imaging (FLIM) and 3D structural illumination microscopy (SIM) showed that all aggregate species displayed highly compact structures with strong affinity between individual fibrils. We also found that aggregates formed by Arctic mutant Aβ42 were more resistant to intracellular degradation than their WT counterparts. Our findings uncover the structural basis of the progression of Arctic mutant Aβ42 aggregation in the cell.

Alzheimer's 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. © 2016 Wiley Periodicals, Inc.

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 Parkinson's and Alzheimer's 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.

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.

We have previously shown that lithium can protect against the polyglutamine toxicity of the Huntington's disease mutation in cell models. Here, we demonstrate for the first time in vivo that lithium can protect against the toxicity caused by aggregate-prone proteins with either polyglutamine or polyalanine expansions in Drosophila. We also show that these protective effects can be partly accounted for by lithium acting through the Wnt/Wg pathway, as a GSK3beta-specific inhibitor and overexpression of dTCF also mediate protective effects. Our data suggest that lithium deserves serious consideration for further studies as a therapeutic for polyglutamine diseases, particularly as it is an established drug that has been used for several decades for chronic treatment of affective disorders.

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.

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.

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.