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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 a 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.

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., Highlights • FUS phase transitions between monomer, liquid droplet, and hydrogel states • FUS mutants induce further phase transition into irreversible fibrillar hydrogels • Irreversible hydrogels sequester RNP cargo and impair RNP granule function • Formation of non-amyloid fibrillar hydrogels provides a compelling causative mechanism for neurodegeneration, Murakami et al. show that FUS transitions between monomer, liquid droplet, and hydrogel states during uptake and release of RNP granule cargo. FUS mutations accelerate transition into fibrillar hydrogels that trap RNP cargo, impair RNP granule function, and cause neurodegeneration.

The aim of this study was to establish epithelial cell lines derived from defined nephron segments. Primary cultures were prepared from dissected proximal S2 segments of the rabbit kidney, and grown in monolayers. Immortalization was observed after nuclear microinjection of the cells with simian virus 40 DNA and resulted in the development of cell lines of epithelial morphology. These cell lines were maintained in culture for at least 24 passages, then cells were frozen. One of the cell lines, the RKPC-2, was selected and further characterized. RKPC-2 cells formed domes on impermeable supports, indicating fluid and solute transport. RKPC-2 cells formed continuous monolayers of low transepithelial resistance on collagen-coated filters. They were able to accumulate tetraethylammonium, an organic cation; however, no significant transcellular transport could be measured. We conclude that this cell line which shows characteristics of epithelial cells has maintained certain properties of intact proximal tubules, in particular the capacity to accumulate organic cations.

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.

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 which 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.

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.

The 42-amino-acid -amyloid (A42) is a critical causative agent in the pathology of Alzheimer’s disease. The hereditary Arctic mutation of A42 (E22G) leads to increased intracellular accumulation of -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 A42 to study the aggregation kinetics of the Arctic A42 mutant peptide and its heterogeneous structural forms. Arctic-mutant peptides assemble and form fibrils at a much faster rate thanWT peptides. We identified five categories of intracellular aggregate— oligomers, single fibrils, fibril bundles, clusters, and aggresomes—that underline the heterogeneity of these A42 aggregates and represent the progression of A42 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 A42 were more resistant to intracellular degradation than theirWT counterparts. Our findings uncover the structural basis of the progression of Arctic mutant A42 aggregation in the cell., This research was funded by Infinitus (China) Company Ltd, and an Advanced Investigator Award (AdG233232) from the European Research Council to AT; G.S.K.S. acknowledges funding from the Wellcome Trust, the UK Medical Research Council (MRC), and Alzheimer Research UK (ARUK).