The role of the VQIVYK peptide in tau protein phosphorylation

Although it remains unclear whether they are related to one another, tau aggregation and phosphorylation are the main pathological hallmarks of the neuronal disorders known as tauopathies. The capacity to aggregate is impaired in a variant of the tau 3R isoform that lacks residues 306–311 (nomenclature for the largest CNS tau isoform) and hence, we have taken advantage of this feature to study how phosphorylation and aggregation may be related as well as the role of this six amino acid peptide (VQIVYK). Through these analyses, we found that the phosphorylation of the tau variant was higher than that of the complete tau protein and that not only the deletion of these residues, but also the interaction of these residues, in tau 3R, with thioflavin-S augmented tau phosphorylation by glycogen synthase kinase 3. In addition, the binding of the peptide containing the residues 306–311 to the whole tau protein provoked an increase in tau phosphorylation. This observation could be physiologically relevant as may suggest that tau–tau interactions, through those residues, facilitate tau phosphorylation. In summary, our data indicate that deletion of residues VQIVYK, in tau protein produces an increase in tau phosphorylation, without tau aggregation, because the VQIVYK peptide, that favors aggregation, is missing. On the other hand, when the whole tau protein interacts with thioflavin-S or the peptide VQIVYK, an increase in both aggregation and phosphorylation occurs.     

Molecular dynamics simulations to investigate the stability and aggregation behaviour of the amyloid-forming peptide VQIVYK from tau protein

The formation of paired helical filaments arising from the short hexapeptide in the third repeat of tau protein, ³⁰⁶VQIVYK³¹¹, is critical for tau polymerisation. The atomic structure of the VQIVYK oligomer has revealed a dry, tightly self-complementing structure between the neighbouring β-sheet layers, termed as ‘steric zipper’. In this study, several molecular dynamics simulations with all-atom explicit water were conducted to investigate the structural stability and aggregation behaviour of the VQIVYK peptide with various sizes and its single alanine replacement mutations. Our results indicate that the van der Waals interaction between side chains of Q2, the π–π stacking interaction between aromatic rings of Y5, and the electrostatic interaction between K6 and the C-terminus play an important role in stabilising the VQIVYK oligomers within the same β-sheet layer, while hydrophobic steric zipper involving V1, I3 and Y5 is responsible for holding the neighbouring β-sheet layers together. The twisted angles of the VQIVYK oligomers were also analysed and shown to be size dependent. The present results not only provide atomic insights into amyloid formation, but are also helpful for designing new or modified capping peptides and inhibitors to prevent fibril formation of the VQIVYK peptide from tau protein.     

Secondary nucleating sequences affect kinetics and thermodynamics of tau aggregation

Tau protein was scanned for highly amyloidogenic sequences in amphiphilic motifs (X)(n)Z, Z(X)(n)Z (n ≥ 2), or (XZ)(n) (n ≥ 2), where X is a hydrophobic residue and Z is a charged or polar residue. N-Acetyl peptides homologous to these sequences were used to study aggregation. Transmission electron microscopy (TEM) showed seven peptides, in addition to well-known primary nucleating sequences Ac(275)VQIINK (AcPHF6*) and Ac(306)VQIVYK (AcPHF6), formed fibers, tubes, ribbons, or rolled sheets. Of the peptides shown by TEM to form amyloid, Ac(10)VME, AcPHF6*, Ac(375)KLTFR, and Ac(393)VYK were found to enhance the fraction of β-structure of AcPHF6 formed at equilibrium, and Ac(375)KLTFR was found to inhibit AcPHF6 and AcPHF6* aggregation kinetics in a dose-dependent manner, consistent with its participation in a hybrid steric zipper model. Single site mutants were generated which transformed predicted amyloidogenic sequences in tau into non-amyloidogenic ones. A M11K mutant had fewer filaments and showed a decrease in aggregation kinetics and an increased lag time compared to wild-type tau, while a F378K mutant showed significantly more filaments. Our results infer that sequences throughout tau, in addition to PHF6 and PHF6*, can seed amyloid formation or affect aggregation kinetics or thermodynamics.     

Loss of intramolecular electrostatic interactions and limited conformational ensemble may promote self‐association of cis–tau peptide

Self‐association of proteins can be triggered by a change in the distribution of the conformational ensemble. Posttranslational modification, such as phosphorylation, can induce a shift in the ensemble of conformations. In the brain of Alzheimer's disease patients, the formation of intra‐cellular neurofibrillary tangles deposition is a result of self‐aggregation of hyper‐phosphorylated tau protein. Biochemical and NMR studies suggest that the cis peptidyl prolyl conformation of a phosphorylated threonine‐proline motif in the tau protein renders tau more prone to aggregation than the trans isomer. However, little is known about the role of peptidyl prolyl cis/trans isomerization in tau aggregation. Here, we show that intra‐molecular electrostatic interactions are better formed in the trans isomer. We explore the conformational landscape of the tau segment containing the phosphorylated‐Thr²³¹‐Pro²³² motif using accelerated molecular dynamics and show that intra‐molecular electrostatic interactions are coupled to the isomeric state of the peptidyl prolyl bond. Our results suggest that the loss of intra‐molecular interactions and the more restricted conformational ensemble of the cis isomer could favor self‐aggregation. The results are consistent with experiments, providing valuable complementary atomistic insights and a hypothetical model for isomer specific aggregation of the tau protein. Proteins 2015; 83:436–444. © 2014 Wiley Periodicals, Inc.     

Influence of temperature on formation of perfect tau fragment fibrils using PRIME20/DMD simulations

We investigate the fibrillization process for amyloid tau fragment peptides (VQIVYK) by applying the discontinuous molecular dynamics method to a system of 48 VQIVYK peptides modeled using a new protein model/force field, PRIME20. The aim of the article is to ascertain which factors are most important in determining whether or not a peptide system forms perfect coherent fibrillar structures. Two different directional criteria are used to determine when a hydrogen bond occurs: the original H‐bond constraints and a parallel preference H‐bond constraint that imparts a slight bias towards the formation of parallel versus antiparallel strands in a β‐sheet. Under the original H‐bond constraints, the resulting fibrillar structures contain a mixture of parallel and antiparallel pairs of strands within each β‐sheet over the whole fibrillization temperature range. Under the parallel preference H‐bond constraints, the β‐sheets within the fibrillar structures are more likely to be parallel and indeed become perfectly parallel, consistent with X‐ray crystallography, at a high temperature slightly below the fibrillization temperature. The high temperature environment encourages the formation of perfect fibril structures by providing enough time and space for peptides to rearrange during the aggregation process. There are two different kinetic mechanisms, template assembly with monomer addition at high temperature and merging/rearrangement without monomer addition at low temperature, which lead to significant differences in the final fibrillar structure. This suggests that the diverse fibril morphologies generally observed in vitro depend more on environmental conditions than has heretofore been appreciated.     

A soluble oligomer of tau associated with fiber formation analyzed by NMR

Alzheimer's disease (AD) is characterized by the intracellular accumulation of the neurofibrillary tangles comprised mainly of the microtubule-associated protein, tau. A critical aspect of understanding tangle formation is to understand the transition of soluble monomeric tau into mature fibrils by characterizing the structure of intermediates along the aggregation pathway. We have carried out multidimensional NMR studies on a C-terminal fragment of human tau (tau (187)) to gain structural insight into the aggregation process. To specifically monitor intermolecular interaction between tau molecules in solution, we combined (15)N- and (14)N-labeled tau, the latter of which was modified with a paramagnetic nitroxide spin label (MTSL). Paramagnetic relaxation enhancement (PRE) of (15)N-tau by interaction with MTSL- (14)N-tau allowed identification of low molecular weight oligomers of tau (187) that formed in response to heparin-induced aggregation. Two regions, VQIINK (280) and VQIVYK (311), were exclusively broadened by MTSL located at varied positions in the tau molecule. We propose that soluble oligomers of tau (187) are generated via intermolecular interactions at these motifs triggered by heparin addition. However, the associated line broadening at these motifs cannot be due to interaction between tau (187) and heparin directly. Instead, these specific interactions necessarily occur between tau molecules and are intermolecular in nature. Our data support the idea that VQIINK (280) and VQIVYK (311) are the major, if not sole, critical regions that directly mediate intermolecular contact between tau molecules during the early phases of aggregation.     

Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimer's disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of beta-structure around the hexapeptide motif PHF6 ((306)VQIVYK(311)) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif ((275)VQIINK(280)) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of beta-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, DeltaK280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for beta-structure around the hexapeptide motifs.     

Prediction of nucleating sequences from amyloidogenic propensities of tau-related peptides

Physical properties, including amyloid morphology, FTIR and CD spectra, enhancement of Congo red absorbance, polymerization rate, critical monomer concentration, free energy of stabilization, hydrophobicity, and the partition coefficient between soluble and amyloid states, were measured for the tau-related peptide Ac-VQIVYK amide (AcPHF6) and its single site mutants Ac-VQIVXK amide (X not equal Cys). Transmission electron microscopy showed that 15 out of the 19 peptides formed amyloid in buffer, with morphologies ranging from straight and twisted filaments to sheets and rolled sheets. Using principal component analysis (PCA), measured properties were treated in a comprehensive manner, and scores along the most significant principal components were used to define individual amino acid amyloidogenic propensities. Quantitative structure-activity modeling (QSAM) showed that residues with greater size and hydrophobicity made the largest contributions to the propensity of peptides to form amyloid. Using individual amino acid propensities, sequences within tau with high amyloid-forming potential were estimated and found to include 226VAVVR230 in the proline-rich region, 275VQIINK280 (PHF6) and 306VQIVYK311 (PHF6) within the microtubule binding region, and 392IVYK395 in the C-tail region of the protein. The results suggest that regions outside the microtubule-binding region may play important roles in tau aggregation kinetics or paired helical filament structure.     

Proteolysis of non-phosphorylated and phosphorylated tau by thrombin

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.     

Effects of cationic vectors complexed with plasmid DNA on the phagosome-lysosome fusion in murine peritoneal macrophages and J744 macrophages

Polyethylenimine (PEI) and cationic polypeptides complexed with plasmid DNA are the most efficient nonviral vectors for gene therapy. It is believed that endocytosis is the major pathway for cell entering by PEI/DNA or cationic peptides/DNA complexes. Effects of plasmid DNA complexed with PEI, poly-L-lysine (PLL), poly-D-lysine (PDL) and polyarginine (PA) on the phagosome-lysosome fusion (P-LF) were studied in murine peritoneal macrophages and J774 macrophages. Cationic polypeptide PLL can be hydrolysed by cellular peptidases, but its stereoisomer, PDL, cannot be split by these enzymes. PEI, PDL, and PA have been shown to inhibit P-LF. PLL showed a low effect on the P-LF. On the basis of these studies, we assume that lysosomotropic agents able to change functions of lysosomes in the cell may affect transfection efficiency and thus be used for gene therapy.     

C-H ... π interplay between Ile308 and Tyr310 residues in the third repeat of microtubule binding domain is indispensable for self-assembly of three- and four-repeat tau

Information on the structural scaffold for tau aggregation is important in developing a method of preventing Alzheimer's disease (AD). Tau contains a microtubule binding domain (MBD) consisting of three or four repeats of 31 and 32 similar residues in its C-terminal half. Although the key event in tau aggregation has been considered to be the formation of β-sheet structures from a short hexapeptide (306)VQIVYK(311) in the third repeat of MBD, its aggregation pathway to filament formation differs between the three- and four-repeated MBDs, owing to the intermolecular and intramolecular disulphide bond formations, respectively. Therefore, the elucidation of a common structural element necessary for the self-assembly of three-/four-repeated full-length tau is an important research subject. Expanding the previous results on the aggregation mechanism of MBD, in this paper, we report that the C-H … π interaction between the Ile308 and Tyr310 side chains in the third repeat of MBD is indispensable for the self-assembly of three-/four-repeated full-length tau, where the interaction provides a conformational seed for triggering the molecular association. On the basis of the aggregation behaviours of a series of MBD and full-length tau mutants, a possible self-association model of tau is proposed and the relationship between the aggregation form (filament or granule) and the association pathway is discussed.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Structure and function of RGD peptides derived from disintegrin proteins

The Arg-Gly-Asp (RGD) sequence serves as the primary recognition site in extracellular matrix proteins, and peptides containing this sequence can mimic the biological activities of matrix proteins. We have initiated structure-function studies of two RGD containing peptides, RGD-5(AGGDD) and cyclic RGD-6(CARGDDC). Assays have shown that cyclic RGD-peptides inhibit platelet aggregation more efficiently than linear ones. NMR data revealed that RGD-5 and RGD-6 have entirely different conformation. RGD-5 has a linear extended structure and RGD-6 has a stable loop conformation. In RGD-5 the guanidinium group of Arg2 and the carboxyl group of Asp4 lie in parallel, whereas the side-chains of Arg3 and Asp5 of RGD-6 are located in different planes, supporting the idea that the stability of the cyclic form derives from the packing of the side chain of the Arg and Asp residues. The structural features of these peptides could provide a basis for designing new drugs against diseases related to platelet aggregation and as cancer antagonists.     

A theoretical study of polymorphism in VQIVYK fibrils

The VQIVYK fragment from the Tau protein, also known as PHF6, is essential for aggregation of Tau into neurofibrillary lesions associated with neurodegenerative diseases. VQIVYK itself forms amyloid fibrils composed of paired β-sheets. Therefore, the full Tau protein and VQIVYK fibrils have been intensively investigated. A central issue in these studies is polymorphism, the ability of a protein to fold into more than one structure. Using all-atom molecular simulations, we generate five stable polymorphs of VQIVYK fibrils, establish their relative free energy with umbrella sampling methods, and identify the side chain interactions that provide stability. The two most stable polymorphs, which have nearly equal free energy, are formed by interdigitation of the mostly hydrophobic VIY “face” sides of the β-sheets. Another stable polymorph is formed by interdigitation of the QVK “back” sides. When we turn to examine structures from cryo-electron microscopy experiments on Tau filaments taken from diseased patients or generated in vitro, we find that the pattern of side chain interactions found in the two most stable face-to-face as well as the back-to-back polymorphs are recapitulated in amyloid structures of the full protein. Thus, our studies suggest that the interactions stabilizing PHF6 fibrils explain the amyloidogenicity of the VQIVYK motif within the full Tau protein and provide justification for the use of VQIVYK fibrils as a test bed for the design of molecules that identify or inhibit amyloid structures.     

Modifications on amphiphilicity and cationicity of unnatural amino acid containing peptides for the improvement of antimicrobial activity against pathogenic bacteria

The widespread natural sources‐derived cationic peptides have been reported to reveal bacterial killing and/or growth‐inhibiting properties. Correspondingly, a number of artificial peptides have been designed to understand antibacterial mechanism of the cationic peptides. These peptides are expected to be an alternative antibiotic against drug‐resistant pathogenic bacteria because major antimicrobial mechanism of cationic peptides involves bacterial membrane disorder, although those availabilities have not been well evaluated. In this study, cationic peptides containing Aib were prepared to evaluate the availability as an antimicrobial agent, especially against representative pathogenic bacteria. Among them, BRBA20, consisting of five repeated Aib‐Arg‐Aib‐Ala sequences, showed strong antibacterial activity against both Gram‐negative and Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus. Additionally, growth of Serratia marcescens and multidrug‐resistant Pseudomonas aeruginosa, known as proteases‐secreting pathogenic bacteria, were also completely inhibited by BRBA20 under 20 µg/ml peptide concentrations. Our results suggested availabilities of Aib‐derived amphiphilicity and protease resistance in the design of artificial antimicrobial peptides. Comparing BRBA20 with BKBA20, it was also concluded that Arg residue is the preferred cationic source than Lys for antimicrobial action of amphiphilic helices. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and evaluation of 1,2,3,4-tetrahydro-1-acridone analogues as potential dual inhibitors for amyloid-beta and tau aggregation

Amyloid-β (Aβ) and tau protein are two crucial hallmarks in Alzheimer’s disease (AD). Their aggregation forms are thought to be toxic to the neurons in the brain. A series of new 1,2,3,4-tetrahydro-1-acridone analogues were designed, synthesized, and evaluated as potential dual inhibitors for Aβ and tau aggregation. In vitro studies showed that compounds 25–30 (20?μM) with N-methylation of the quinolone ring effectively inhibited Aβ_1-42 aggregation by 84.7%–99.5% and tau aggregation by 71.2%–101.8%. Their structure-activity relationships are discussed. In particular, 30 could permeate the blood-brain barrier, bind to Aβ_1-42 and tau, inhibit Aβ_1-42 β-sheets formation, and prevent tau aggregation in living cells.     

Novel conformation-sensitive antibodies specific to three- and four-repeat tau

Two types of tau isoform, three- and four-repeat tau, are found in neurofibrillary tangles--a pathological hallmark of tauopathies. Which isoform is deposited in the affected tissues depends on the tauopathy. To study how and which tau isoforms contribute to neuronal degeneration, we have developed and characterized two novel conformation-sensitive antibodies, T3R and T4R. Two closely related synthetic peptides, PGGGKVQIVYK and PGGGSVQIVYK, respectively, were designed as antigens. The isoform-specific residues, (305)K in three-repeat tau or (305)S in four-repeat tau, and the PHF6 motif (VQIVYK) were identified as critical sequences. Despite the high similarity of the antigens, there was no cross-reactivity between T3R and T4R. Furthermore, T3R and T4R showed reduced binding to the thioflavin-positive beta-structural form of their target. These features may enable these antibodies to act as novel indicators that allow us to observe and evaluate conformational changes in each distinct isoform of tau.     

EFhd2 is a novel amyloid protein associated with pathological tau in Alzheimer's disease

EFhd2 is a conserved calcium‐binding protein, abundant within the central nervous system. Previous studies identified EFhd2 associated with pathological forms of tau proteins in the tauopathy mouse model JNPL3, which expresses the human tau^P301L mutant. This association was validated in human tauopathies, such as Alzheimer's disease (AD). However, the role that EFhd2 may play in tauopathies is still unknown. Here, we show that EFhd2 formed amyloid structures in vitro, a capability that is reduced by calcium ions. Electron microscopy (EM) analyses demonstrated that recombinant EFhd2 formed filamentous structures. EM analyses of sarkosyl‐insoluble fractions derived from human AD brains also indicated that EFhd2 co‐localizes with aggregated tau proteins and formed granular structures. Immunohistological analyses of brain slices demonstrated that EFhd2 co‐localizes with pathological tau proteins in AD brains, confirming the co‐aggregation of EFhd2 and pathological tau. Furthermore, EFhd2's coiled‐coil domain mediated its self‐oligomerization in vitro and its association with tau proteins in JNPL3 mouse brain extracts. The results demonstrate that EFhd2 is a novel amyloid protein associated with pathological tau proteins in AD brain and that calcium binding may regulate the formation of EFhd2's amyloid structures. Hence, EFhd2 may play an important role in the pathobiology of tau‐mediated neurodegeneration.     

Amyloid‐like aggregation of designer bolaamphiphilic peptides: Effect of hydrophobic section and hydrophilic heads

Amyloid‐like aggregation of natural proteins or polypeptides is an important process involved in many human diseases as well as some normal biological functions. Plenty of works have been done on this ubiquitous phenomenon, but the molecular mechanism of amyloid‐like aggregation has not been fully understood yet. In this study, we showed that a series of designer bolaamphiphilic peptides could undergo amyloid‐like aggregation even though they didn't possess typical β‐sheet secondary structure. Through systematic amino acid substitution, we found that for the self‐assembling ability, the number and species of amino acid in hydrophobic section could be variable as long as enough hydrophobic interaction is provided, while different polar amino acids as the hydrophilic heads could change the self‐assembling nanostructures with their aggregating behaviors affected by pH value change. Based on these results, novel self‐assembling models and aggregating mechanisms were proposed, which might provide new insight into the molecular basis of amyloid‐like aggregation.     

Tau protein liquid–liquid phase separation can initiate tau aggregation

The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid–liquid phase separation (LLPS) under cellular conditions and that phase‐separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho‐tau isolated from human Alzheimer brain. Droplet‐like tau can also be observed in neurons and other cells. We found that tau droplets become gel‐like in minutes, and over days start to spontaneously form thioflavin‐S‐positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases.