SummaryThe 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.
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.
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.
Reduced protein homeostasis leading to increased protein instability is a common molecular feature of aging, but it remains unclear whether this is a cause or consequence of the aging process. In neurodegenerative diseases and other amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological aggregates in different tissues. More recently, widespread protein aggregation has been described during normal aging. Until now, an extensive characterization of the nature of age-dependent protein aggregation has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and have an amyloid-like structure resembling that of protein aggregates observed in disease. We then demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in C. elegans. Together, these findings imply that amyloid-like aggregates contribute to the aging process and therefore could be important targets for strategies designed to maintain physiological functions in the late stages of life.
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.T he proliferation of α-synuclein (AS) aggregates (1-6), including the existence of distinct "prion-like" strains (7, 8) as well as their spatial propagation throughout the brain (9), has been proposed to occur in the brains of patients suffering from Parkinson's disease, but their links to pathology and to neuronal death have remained elusive (10-13). In this study, we use optical nanoscopy (14-18) to observe neurons directly and to assay AS internalization, as well as fibril-induced templating reactions involving endogenous AS at the molecular level. We further correlate this information with toxic phenotypes.We have previously shown that the presence of AS short fibrils (seed fibrils or seeds) preformed in vitro (Materials and Methods and Fig. 1 and Fig. S1) favors elongation reactions over spontaneous nucleation (term used for seed-independent aggregation hereafter) in vitro (18). Furthermore, the seed fibrils were found to display highly inhomogeneous growth kinetics, with a significant fraction showing little or no growth at all (18). Here, we use two-color direct stochastic optical reconstruction microscopy (dSTORM), a superresolution imaging method (14), to investigate how such processes may be modified in the cellular environment. The results show the potential of this technique for studying the mechanisms of aggregation events in vivo and provide evidence for the neuroprotective role of reducing the concentration of free AS in the cellular environment. Results and DiscussionExternally Added Seed Fibrils of AS Act as Templates for Exogenously Added Monomeric AS and Prevent Its Nucleation. We first incubated neuronal cells with AS seed fibrils [50 nM, 5% covalently labeled with Alexa Fluor 568 (AF568), green] and AS monomers [500 nM, 10% covalently labeled with Alexa Fluor 647 (AF647), red], either each individually or both in sequence. Either seed fibrils and...
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