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Phase separation is an important process in biology associated with formation of membraneless organelles but possibly related to the emergence of solid inclusions. TDP-43 is a largely studied paradigmatic case, as it forms neuronal cytoplasmic inclusions in neurodegenerative diseases and is an essential component of many membraneless organelles. Here, we review the physicochemical fundamentals of liquid–liquid phase separation (LLPS) of TDP-43 and its fragments in vitro, showing that full-length TDP-43 requires RNA or chaperones to form stable liquid droplets. We describe TDP-43-containing membraneless organelles and the debate on whether these assemblies represent reservoirs for pathological solid inclusion formation.

The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson’s disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species. The self-assembly of α-synuclein (αS) is a pathological feature of Parkinson’s disease. The αS species responsible for neuronal damage are not well characterized. Here, the authors show that αS fibrils release soluble prefibrillar oligomeric species responsible for neurotoxicity in vitro.

Background Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-TDP), amyotrophic lateral sclerosis (ALS) and limbic-predominant age-related TDP-43 encephalopathy (LATE) are associated with deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons. One complexity of this process lies in the ability of TDP-43 to form liquid-phase membraneless organelles in cells. Previous work has shown that the recombinant, purified, prion-like domain (PrLD) forms liquid droplets in vitro, but the behaviour of the complementary fragment is uncertain. Methods We have purified such a construct without the PrLD (PrLD-less TDP-43) and have induced its phase separation using a solution-jump method and an array of biophysical techniques to study the morphology, state of matter and structure of the TDP-43 assemblies. Results The fluorescent TMR-labelled protein construct, imaged using confocal fluorescence, formed rapidly (< 1 min) round, homogeneous and 0.5–1.0 µm wide assemblies which then coalesced into larger, yet round, species. When labelled with AlexaFluor488, they initially exhibited fluorescence recovery after photobleaching (FRAP), showing a liquid behaviour distinct from full-length TDP-43 and similar to PrLD. The protein molecules did not undergo major structural changes, as determined with circular dichroism and intrinsic fluorescence spectroscopies. This process had a pH and salt dependence distinct from those of full-length TDP-43 and its PrLD, which can be rationalized on the grounds of electrostatic forces. Conclusions Similarly to PrLD, PrLD-less TDP-43 forms liquid droplets in vitro through liquid–liquid phase separation (LLPS), unlike the full-length protein that rather undergoes liquid–solid phase separation (LSPS). These results offer a rationale of the complex electrostatic forces governing phase separation of full-length TDP-43 and its fragments. On the one hand, PrLD-less TDP-43 has a low pI and oppositively charged domains, and LLPS is inhibited by salts, which attenuate inter-domain electrostatic attractions. On the other hand, PrLD is positively charged due to a high isoionic point (pI) and LLPS is therefore promoted by salts and pH increases as they both reduce electrostatic repulsions. By contrast, full-length TDP-43 undergoes LSPS most favourably at its pI, with positive and negative salt dependences at lower and higher pH, respectively, depending on whether repulsive or attractive forces dominate, respectively.

Background Amyloid-β 42 (Aβ 42 ) aggregation consists of a complex chain of nucleation events producing soluble oligomeric intermediates, which are considered the major neurotoxic agents in Alzheimer’s disease (AD). Cerebral lesions in the brain of AD patients start to develop 20 years before symptom onset; however, no preventive strategies, effective treatments, or specific and sensitive diagnostic tests to identify people with early-stage AD are currently available. In addition, the isolation and characterisation of neurotoxic Aβ 42 oligomers are particularly difficult because of their transient and heterogeneous nature. To overcome this challenge, a rationally designed method generated a single-domain antibody (sdAb), named DesAb-O, targeting Aβ 42 oligomers. Methods We investigated the ability of DesAb-O to selectively detect preformed Aβ 42 oligomers both in vitro and in cultured neuronal cells, by using dot-blot, ELISA immunoassay and super-resolution STED microscopy, and to counteract the toxicity induced by the oligomers, monitoring their interaction with neuronal membrane and the resulting mitochondrial impairment. We then applied this approach to CSF samples (CSFs) from AD patients as compared to age-matched control subjects. Results DesAb-O was found to selectively detect synthetic Aβ 42 oligomers both in vitro and in cultured cells, and to neutralise their associated neuronal dysfunction. DesAb-O can also identify Aβ 42 oligomers present in the CSFs of AD patients with respect to healthy individuals, and completely prevent cell dysfunction induced by the administration of CSFs to neuronal cells. Conclusions Taken together, our data indicate a promising method for the improvement of an early diagnosis of AD and for the generation of novel therapeutic approaches based on sdAbs for the treatment of AD and other devastating neurodegenerative conditions.

α-Synuclein (αS) is an intrinsically disordered and highly dynamic protein involved in dopamine release at presynaptic terminals. The abnormal aggregation of αS as mature fibrils into intraneuronal inclusion bodies is directly linked to Parkinson’s disease. Increasing experimental evidence suggests that soluble oligomers formed early during the aggregation process are the most cytotoxic forms of αS. This study investigated the uptake by neuronal cells of pathologically relevant αS oligomers and fibrils exploiting a range of conformation-sensitive antibodies, and the super-resolution stimulated emission depletion (STED) microscopy. We found that prefibrillar oligomers promptly penetrate neuronal membranes, thus resulting in cell dysfunction. By contrast, fibril docking to the phospholipid bilayer is accompanied by αS conformational changes with a progressive release of A11-reactive oligomers, which can enter into the neurons and trigger cell impairment. Our data provide important evidence on the role of αS fibrils as a source of harmful oligomers, which resemble the intermediate conformers formed de novo during aggregation, underling the dynamic and reversible nature of protein aggregates responsible for α-synucleinopathies.

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-β peptide (Aβ) and of α-synuclein (αS), which are associated with Alzheimer’s and Parkinson’s diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aβ and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aβ and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer’s and Parkinson’s diseases.

Background The aggregation of α-Synuclein (αS) into amyloid fibrils and their deposition in intraneuronal Lewy bodies are hallmark features of Parkinson's disease (PD) and other synucleinopathies. Among the molecular players implicated in αS toxicity, the cellular prion protein (PrP C ) has emerged as a potential modulator of αS-neuron interactions. Results Using confocal microscopy, colocalization analysis and both siRNA-induced PrP C silencing and antibody-based blockade, we investigated the contribution of PrP C to αS-induced neurotoxicity in human iPSC-derived dopaminergic neurons, primary rat cortical neurons and human SH-SY5Y neuroblastoma cells. We show that PrP C facilitated the early recruitment of αS prefibrillar type B* oligomers (OB*) and short fibrils (SF) to neuronal membranes, enhancing αS-induced Ca 2+ influx and membrane permeabilization. However, PrP C levels remained unchanged following prolonged exposure with OB* and SF, suggesting no feedback modulation of PrP C expression. While PrP C blockade partially inhibited the release of toxic soluble oligomers from αS fibrils, downstream cell death was only marginally reduced, indicating a limited contribution of PrP C to the final neurotoxic outcome. By contrast, extracellular Ca 2+ emerged as a major driver of αS toxicity, directly promoting the membrane recruitment, internalization and cytotoxic effects of αS aggregates. Conclusions Collectively, our findings indicate that while PrP C facilitates early events in αS aggregate interaction with neurons, the sustained neurotoxicity induced by αS prefibrillar oligomers and fibrils is predominantly mediated by extracellular Ca 2+ . This promotes aggregate-membrane interactions, membrane permeabilization, and intracellular Ca 2+ dyshomeostasis, thereby establishing a vicious cycle of neuronal dysfunction and death.

Protein misfolding is a general hallmark of protein deposition diseases, such as Alzheimer’s disease or Parkinson’s disease, in which different types of aggregated species (oligomers, protofibrils and fibrils) are generated by the cells. Despite widespread interest, the relationship between oligomers and fibrils in the aggregation process and spreading remains elusive. A large variety of experimental evidences supported the idea that soluble oligomeric species of different proteins might be more toxic than the larger fibrillar forms. Furthermore, the lack of correlation between the presence of the typical pathological inclusions and disease sustained this debate. However, recent data show that the β-sheet core of the α-Synuclein (αSyn) fibrils is unable to establish persistent interactions with the lipid bilayers, but they can release oligomeric species responsible for an immediate dysfunction of the recipient neurons. Reversibly, such oligomeric species could also contribute to pathogenesis via neuron-to-neuron spreading by their direct cell-to-cell transfer or by generating new fibrils, following their neuronal uptake. In this Review, we discuss the various mechanisms of cellular dysfunction caused by αSyn, including oligomer toxicity, fibril toxicity and fibril spreading.