TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

A characteristic of progressive neurodegenerative tauopathies is the accumulation of insoluble neurofibrillary tangles (NFTs) composed of hyperphosphorylated fibrillar tau protein (f.tau) (microtubule-associated protein tau [MAPT]) in affected neurons (1). Fibrillization can be recapitulated in vitro by mixing recombinant human tau with polyanionic compounds such as heparin, arachidonic acid, or RNA (2, 3). Through nonspecific electrostatic interaction with the tau repeat domain, RNA may function as a physiological inducer of tau fibrillization (4, 5). However, f.tau does not appear to be the primary neurotoxic driver of toxicity in tauopathies (6, 7). Mounting evidence suggests that toxicity mainly arises from oligomeric tau (o.tau) species (8–11), which are thought to be temporal intermediate tau units in the polymerization of f.tau (12, 13).Phase separation has emerged as a fundamentally important process in biology (14, 15). Nonmembrane bound protein condensates form in response to cellular signals and allow for dynamic compartmentalization of biomolecules for the completion of defined biochemical reactions. Recently, tau was shown to undergo liquid–liquid phase separation (LLPS) in the presence of molecular crowding agents (16). The low sequence complexity of tau generates intrinsically disordered regions (IDRs), which are characteristic of proteins that undergo phase separation (17, 18). Initially, phase separation produces droplets of dynamic liquid tau (l.tau) that with time gelates into vitrified tau (v.tau) (19). Condensates of tau may be a necessary stage in recruiting and concentrating tubulin (∼10-fold higher than outside tau droplets) for the purpose of nucleating microtubules (16). Tau also coacervates with RNA into liquid droplets in vitro (20, 21) and with RNA binding proteins (RBPs) in cells in response to stress (22, 23). More recent work showed that PEG-induced LLPS can generate tau oligomers involving N-terminal exposure in the absence of fibril formation (24). However, prior work leaves unknown whether phase separation induced by RNA or RBPs plays a role in tau pathology and tau toxicity. In Alzheimer’s disease (AD) models, tau interacts with RBPs in a manner that induces pathogenic alterations to tau (25, 26). Interaction of tau with TIA1, a stress granule (SG) nucleating RBP (27), is necessary for tau-dependent neurodegeneration in PS19 transgenic mice (8). Survival of and behavioral deficits in tau transgenic mice correlated with levels of TIA1-dependent oligomeric tau. We subsequently showed that TIA1 is required for the propagation of oligomeric tau (but not fibrillar tau) across the brains of young PS19 mice, resulting in o.tau-dependent neurodegeneration (9). This raises the possibility that interaction with RBPs facilitates the phase separation of tau and the production of toxic tau species.The kinetics of phase separation are governed by determinants that affect intermolecular interactions (28) and the sufficient availability of interaction sites (a biomolecule’s “valence”) on each particle within a connected system (29). For RBPs (and tau), phase separation is dependent on temperature, pH, and on concentration of RNA, salt, and crowding agent following the Flory–Huggins theory of polymer solutions (30, 31). This theory describes the combined energetics of protein–protein, RNA–RNA, protein–RNA, protein–solute, and RNA–solute interactions within the system. As such, phase separation occurs when homo- and heterogeneous molecular interactions between tau and other biomolecules (such as RNA) offer sufficient gain in enthalpy to surmount the loss of system entropy.Here we show an essential role for RBPs in directing tau phase separation and the type of tau pathology that accumulates. In the presence of RNA and the RNA binding protein TIA1, phase separation of tau occurs at physiological concentrations. Furthermore, TIA1 blocks the RNA-dependent conversion of o.tau into f.tau in vitro, leading to accumulation of neurotoxic o.tau species. Our data strongly suggest that direct interaction with TIA1 promotes phase separation of tau and regulates the formation of toxic oligomeric tau.     

Widespread occurrence of the droplet state of proteins in the human proteome

A wide range of proteins have been reported to condensate into a dense liquid phase, forming a reversible droplet state. Failure in the control of the droplet state can lead to the formation of the more stable amyloid state, which is often disease-related. These observations prompt the question of how many proteins can undergo liquid–liquid phase separation. Here, in order to address this problem, we discuss the biophysical principles underlying the droplet state of proteins by analyzing current evidence for droplet-driver and droplet-client proteins. Based on the concept that the droplet state is stabilized by the large conformational entropy associated with nonspecific side-chain interactions, we develop the FuzDrop method to predict droplet-promoting regions and proteins, which can spontaneously phase separate. We use this approach to carry out a proteome-level study to rank proteins according to their propensity to form the droplet state, spontaneously or via partner interactions. Our results lead to the conclusion that the droplet state could be, at least transiently, accessible to most proteins under conditions found in the cellular environment.

It has been recently observed that proteins can self-assemble through a liquid–liquid phase separation (LLPS) process into a dense liquid phase, while maintaining at least in part their functional native states (1–4). These liquid-like assemblies of complex compositions are often referred to as biomolecular condensates or membraneless organelles (1–4). Here, we refer to these dynamic and reversible condensates as droplets, in order to distinguish them from irreversible amyloids. Droplets can concentrate cellular components to perform efficiently a variety of different functions, with an increasing number of biological roles being discovered (1–4).In this work, we investigate whether liquid–liquid phase separation can be expected to be a proteome-wide phenomenon. In this view, the condensation of proteins from the native state to the amyloid state may quite generally proceed through an intermediate dense liquid phase, which is typically metastable (5) (Fig. 1). Different proteins may have different propensities to remain in this metastable phase, depending in particular on the free energy barrier between the droplet and amyloid states (Fig. 1). This type of liquid–liquid phase separation is indeed typical of condensation phenomena (1, 6), and sometimes is referred to as the Ostwald step rule (7). One may think that for most proteins the free energy barrier between the droplet and fibrillar states is low, and therefore the droplet state cannot be readily observed (Fig. 1). Indeed, this state may be difficult to detect due to a variety of reasons, including because experimental methods to probe its formation, in particular high-throughput ones, are still under development (8). Furthermore, our current understanding of the interactions that stabilize the metastable dense liquid phase is still incomplete.Open in a separate windowFig. 1.Liquid–liquid phase separation could be expected to be a proteome-wide phenomenon. Proteins that undergo condensation convert from the native state to the amyloid state through a dense liquid state (the droplet state). The stability of these different states (the minima in the free energy), as well as the conversion rates between them (the barriers in the free energy), is different for different proteins. For most proteins under cellular conditions, the native and droplet states could be expected to be metastable (56), being kinetically trapped by a free energy barrier (ΔG) between the droplet and fibrillar states. Proteins that can be observed in the droplet state tend to have a high free energy barrier (LLPS; green) while the other ones tend to have a low free energy barrier compared with the thermal energy (non-LLPS; orange). For certain proteins the droplet state is functional, and it is stabilized by extrinsic factors, such as RNA and posttranslational modifications.Native and amyloid states are stabilized by specific interactions including hydrogen bonds, ionic interactions, and van der Waals contacts typical of ordered states and enthalpic in nature (9, 10). By contrast, in droplets, transient short-range aromatic cation–π and π–π, dipole–dipole, and electrostatic and hydrophobic interactions have been observed, providing low-specificity, weak-affinity contacts characteristic of disordered states (11–16). These observations have led to a series of prediction methods (11, 13, 17–19), which focused on specific side-chain interactions. The redundancy and multivalency of the interacting elements (20) suggest that conformational entropy is a driving force of the condensation (21), also including main-chain contributions. Indeed, proteins exhibiting many binding configurations with a specific partner are often capable of forming droplets (22).Here, we exploit the observation that many proteins exhibit high conformational entropy upon binding, which can be predicted from their amino acid sequences (23). Based on this result, we develop the FuzDrop method to predict the droplet-promoting propensity of proteins and their droplet-promoting profiles based on the conformational entropy of their free states and the binding entropy. Using this method, we identify a list of “droplet-driving” proteins, which are predicted to undergo spontaneous liquid–liquid phase separation under physiological conditions, and estimate that they comprise about 40% of the human proteome. In addition, we also predict that about 80% of the proteins are “droplet clients,” characterized by short droplet-promoting regions in their sequences, which facilitate condensation via interactions with suitable partners. Taken together, our results indicate that protein phase separation is a proteome-wide phenomenon.     

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left–right symmetry breaking

Proper left–right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left–right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left–right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin–dependent manner. Altogether, we conclude that CYK-1/Formin–dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left–right symmetry breaking in the nematode worm.

The emergence of left–right asymmetry is essential for normal animal development and, in the majority of animal species, one type of handedness is dominant (1). The actin cytoskeleton plays an instrumental role in establishing the left–right asymmetric body plan of invertebrates like fruit flies (2–6), nematodes (7–11), and pond snails (12–15). Moreover, an increasing number of studies showed that vertebrate left–right patterning also depends on a functional actomyosin cytoskeleton (13, 16–22). Actomyosin-dependent chiral behavior has even been reported in isolated cells (23–28) and such cell-intrinsic chirality has been shown to promote left–right asymmetric morphogenesis of tissues (29, 30), organs (21, 31), and entire embryonic body plans (12, 13, 32, 33). Active force generation in the actin cytoskeleton is responsible for shaping cells and tissues during embryo morphogenesis. Torques are rotational forces with a given handedness and it has been proposed that in plane, active torque generation in the actin cytoskeleton drives chiral morphogenesis (7, 8, 34, 35).What could be the molecular origin of these active torques? The actomyosin cytoskeleton consists of actin filaments, actin-binding proteins, and Myosin motors. Actin filaments are polar polymers with a right-handed helical pitch and are therefore chiral themselves (36, 37). Due to the right-handed pitch of filamentous actin, Myosin motors can rotate actin filaments along their long axis while pulling on them (33, 38–42). Similarly, when physically constrained, members of the Formin family rotate actin filaments along their long axis while elongating them (43). In both cases the handedness of this rotation is determined by the helical nature of the actin polymer. From this it follows that both Formins and Myosins are a potential source of molecular torque generation that could drive cellular and organismal chirality. Indeed, chiral processes across different length scales, and across species, are dependent on Myosins (19), Formins (13–15, 26), or both (7, 8, 21, 44). It is, however, unclear how Formins and Myosins contribute to active torque generation and the emergence chiral processes in developing embryos.In our previous work we showed that the actomyosin cortex of some Caenorhabditis elegans embryonic blastomeres undergoes chiral counterrotations with consistent handedness (7, 35). These chiral actomyosin flows can be recapitulated using active chiral fluid theory that describes the actomyosin layer as a thin-film, active gel that generates active torques (7, 45, 46). Chiral counterrotating cortical flows reorient the cell division axis, which is essential for normal left–right symmetry breaking (7, 47). Moreover, cortical counterrotations with the same handedness have been observed in Xenopus one-cell embryos (32), suggesting that chiral counterrotations are conserved among distant species. Chiral counterrotating actomyosin flow in C. elegans blastomeres is driven by RhoA signaling and is dependent on Non-Muscle Myosin II motor proteins (7). Moreover, the Formin CYK-1 has been implicated in actomyosin flow chirality during early polarization of the zygote as well as during the first cytokinesis (48, 49). Despite having identified a role for Myosins and Formins, the underlying mechanism by which active torques are generated remains elusive.Here we show that the Diaphanous-like Formin, CYK-1/Formin, is a critical determinant for the emergence of actomyosin flow chirality, while Non-Muscle Myosin II (NMY-2) plays a permissive role. Our results show that cortical CYK-1/Formin is recruited by active RhoA signaling foci and promotes active torque generation, which in turn tends to locally rotate the actomyosin cortex clockwise. In the highly connected actomyosin meshwork, a gradient of these active torques drives the emergence of chiral counterrotating cortical flows with uniform handedness, which is essential for proper left–right symmetry breaking. Together, these results provide mechanistic insight into how Formin-dependent torque generation drives cellular and organismal left–right symmetry breaking.     

Characterization of the strain-rate–dependent mechanical response of single cell–cell junctions

Cell–cell adhesions are often subjected to mechanical strains of different rates and magnitudes in normal tissue function. However, the rate-dependent mechanical behavior of individual cell–cell adhesions has not been fully characterized due to the lack of proper experimental techniques and therefore remains elusive. This is particularly true under large strain conditions, which may potentially lead to cell–cell adhesion dissociation and ultimately tissue fracture. In this study, we designed and fabricated a single-cell adhesion micro tensile tester (SCAµTT) using two-photon polymerization and performed displacement-controlled tensile tests of individual pairs of adherent epithelial cells with a mature cell–cell adhesion. Straining the cytoskeleton–cell adhesion complex system reveals a passive shear-thinning viscoelastic behavior and a rate-dependent active stress-relaxation mechanism mediated by cytoskeleton growth. Under low strain rates, stress relaxation mediated by the cytoskeleton can effectively relax junctional stress buildup and prevent adhesion bond rupture. Cadherin bond dissociation also exhibits rate-dependent strengthening, in which increased strain rate results in elevated stress levels at which cadherin bonds fail. This bond dissociation becomes a synchronized catastrophic event that leads to junction fracture at high strain rates. Even at high strain rates, a single cell–cell junction displays a remarkable tensile strength to sustain a strain as much as 200% before complete junction rupture. Collectively, the platform and the biophysical understandings in this study are expected to build a foundation for the mechanistic investigation of the adaptive viscoelasticity of the cell–cell junction.

Adhesive organelles between neighboring epithelial cells form an integrated network as the foundation of complex tissues (1). As part of normal physiology, this integrated network is constantly exposed to mechanical stress and strain, which is essential to normal cellular activities, such as proliferation (2–4), migration (5, 6), differentiation (7), and gene regulation (7, 8) associated with a diverse set of functions in tissue morphogenesis (9–11) and wound healing (9). A host of developmental defects or clinical pathologies in the form of compromised cell–cell associations will arise when cells fail to withstand external mechanical stress due to genetic mutations or pathological perturbations (12, 13). Indeed, since the mechanical stresses are mainly sustained by the intercellular junctions, which may represent the weakest link and limit the stress tolerance within the cytoskeleton network of a cell sheet, mutations or disease-induced changes in junction molecules and components in adherens junctions and desmosomes lead to cell layer fracture and tissue fragility, which exacerbate the pathological conditions (14–17). This clinical relevance gives rise to the importance of understanding biophysical transformations of the cell–cell adhesion interface when cells are subjected to mechanical loads.As part of their normal functions, cells often experience strains of tens to a few hundred percent at strain rates of 10⁻⁴ to 1 s⁻¹ (18–21). For instance, embryonic epithelia are subjected to strain rates in the range of 10⁻⁴ to 10⁻³ s⁻¹ during normal embryogenesis (22). Strain rates higher than 0.1 s⁻¹ are often experienced by adult epithelia during various normal physiological functions (21, 23, 24), such as breathing motions in the lung (1 to 10 s⁻¹) (25), cardiac pulses in the heart (1 to 6.5 s⁻¹) (20), peristaltic movements in the gut (0.4 to 1.5 s⁻¹), and normal stretching of the skin (0.1 to 5 s⁻¹). Cells have different mechanisms to dissipate the internal stress produced by external strain to avoid fracture, often via cytoskeleton remodeling and cell–cell adhesion enhancement (26, 27). These coping mechanisms may have different characteristic timescales. Cytoskeleton remodeling can dissipate mechanical stress promptly due to its viscoelastic nature and the actomyosin-mediated cell contractility (17, 28–32). Adhesion enhancement at the cell–cell contact is more complex in terms of timescale. Load-induced cell–cell adhesion strengthening has been shown via the increase in the number of adhesion complexes (33–35) or by the clustering of adhesion complexes (36–39), which occurs on a timescale ranging from a few minutes up to a few hours after cells experience an initial load (28). External load on the cell–cell contact also results in a prolonged cell–cell adhesion dissociation time (40, 41), suggesting cadherin bonds may transition to catch bonds under certain loading conditions (42, 43), which can occur within seconds (44). With the increase in cellular tension, failure to dissipate the stress within the cell layer at a rate faster than the accumulation rate will inevitably lead to the fracture of the cell layer (45). Indeed, epithelial fracture often aggravates the pathological outcomes in several diseases, such as acute lung injuries (46), skin disorders (47), and development defects (48). It is generally accepted that stress accumulation in the cytoskeleton network (49, 50) and potentially in the cytoplasm is strain-rate–dependent (51). However, to date, there is a lack of understanding about the rate-dependent behavior of cell–cell adhesions, particularly about which of the stress-relaxation mechanisms are at play across the spectrum of strain rates. In addition, it remains unclear how the stress relaxation interplays with adhesion enhancement under large strains, especially at high strain rates which may lead to fracture, that is, a complete separation of mature cell–cell adhesions under a tensile load (45, 52, 53). Yet, currently, there is a lack of quantitative technology that enables the investigation of these mechanobiological processes in a precisely controlled manner. This is especially true at high strain rates.To delineate this mechanical behavior, the cleanest characterization method is to directly measure stress dynamics at a single mature cell–cell adhesion interface. Specifically, just as a monolayer cell sheet is a reduction from three-dimensional (3D) tissue, a single cell–cell adhesion interface, as a reduction from a monolayer system, represents the smallest unit to study the rheological behavior of cellular junctions. The mechanistic understanding uncovered with this single unit will inform cellular adaptations to a more complex stress microenvironment in vivo and in vitro, in healthy and diseased conditions. To this end, we developed a single-cell adhesion micro tensile tester (SCAµTT) platform based on nanofabricated polymeric structures using two-photon polymerization (TPP). This platform allows in situ investigation of stress–strain characteristics of a mature cell–cell junction through defined strains and strain rates. With SCAµTT, we reveal some interesting biophysical phenomena at the single cell–cell junction that were previously not possible to observe using existing techniques. We show that cytoskeleton growth can effectively relax intercellular stress between an adherent cell pair in a strain-rate–dependent manner. Along with cadherin-clustering–induced bond strengthening, it prevents failure to occur at low strain rates. At high strain rates, insufficient relaxation leads to stress accumulation, which results in cell–cell junction rupture. We show that a remarkably large strain can be sustained before junction rupture (>200%), even at a strain rate as high as 0.5 s⁻¹. Collectively, the rate-dependent mechanical characterization of the cell–cell junction builds the foundation for an improved mechanistic understanding of junction adaptation to an external load and potentially the spatiotemporal coordination of participating molecules at the cell–cell junction.     

Ion mobility–mass spectrometry reveals the role of peripheral myelin protein dimers in peripheral neuropathy

Peripheral myelin protein (PMP22) is an integral membrane protein that traffics inefficiently even in wild-type (WT) form, with only 20% of the WT protein reaching its final plasma membrane destination in myelinating Schwann cells. Misfolding of PMP22 has been identified as a key factor in multiple peripheral neuropathies, including Charcot-Marie-Tooth disease and Dejerine–Sottas syndrome. While biophysical analyses of disease-associated PMP22 mutants show altered protein stabilities, leading to reduced surface trafficking and loss of PMP22 function, it remains unclear how destabilization of PMP22 mutations causes mistrafficking. Here, native ion mobility–mass spectrometry (IM-MS) is used to compare the gas phase stabilities and abundances for an array of mutant PM22 complexes. We find key differences in the PMP22 mutant stabilities and propensities to form homodimeric complexes. Of particular note, we observe that severely destabilized forms of PMP22 exhibit a higher propensity to dimerize than WT PMP22. Furthermore, we employ lipid raft–mimicking SCOR bicelles to study PMP22 mutants, and find that the differences in dimer abundances are amplified in this medium when compared to micelle-based data, with disease mutants exhibiting up to 4 times more dimer than WT when liberated from SCOR bicelles. We combine our findings with previous cellular data to propose that the formation of PMP22 dimers from destabilized monomers is a key element of PMP22 mistrafficking.

The misfolding of membrane proteins is implicated in the mechanisms of multiple debilitating diseases such as cystic fibrosis and retinitis pigmentosa (1–4). Specific membrane protein mutations are often associated with disease states, with variant forms exhibiting altered stability and cellular trafficking (5). Unfortunately, due to the challenges associated with preparing and handling pure, highly concentrated membrane protein samples, detailed structural information on such targets is often lacking, especially for disease mutant forms. Furthermore, as some membrane proteins associated with misfolding-based diseases have hundreds of mutations of interest (3), there is a clear need for high-throughput methods to assess disease mutation-induced changes in membrane protein stability and structure.Peripheral myelin protein 22 (PMP22) is such a membrane protein, for which misfolding and trafficking of mutant variants have been implicated in disease (6). PMP22 is a tetra-span integral membrane glycoprotein predominately expressed in Schwann cells, which are the principal glial cells of the peripheral nervous system (PNS), where they produce myelin (7–9). In addition to accounting for ∼5% of the protein found in the myelin sheath surrounding PNS nerve axons, PMP22 is thought to regulate intracellular Ca²⁺ levels (10), apoptosis (11), linkage of the actin cytoskeleton with lipid rafts (12), formation of epithelial intercellular junctions (13), myelin formation (14), lipid metabolism, and cholesterol trafficking (15). Dysregulation and misfolding of PMP22 has been identified as a key factor in multiple neurodegenerative disorders, such as Charcot-Marie-Tooth disease types 1A and E, as well as Dejerine–Sottas syndrome (6, 16–18). Like a number of other disease-linked membrane proteins (19), the trafficking of PMP22 is known to be inefficient, with only 20% of the wild-type (WT) protein reaching its final plasma membrane destination in Schwann cells (16, 20). Previously, it has been shown through a range of biophysical analyses that disease-associated PMP22 mutations lower thermodynamic protein stability as the root cause of reduced trafficking and loss of protein function; however, the mechanism by which destabilization of PMP22 causes mistrafficking is still not well understood (6). Additionally, a high-resolution structure of PMP22 has not yet been published.Native mass spectrometry (MS) has recently been demonstrated to overcome sample purity and concentration barriers to reveal critical details of membrane protein structure and function (21–23). Through the use of nano-electrospray (nESI), intact membrane proteins are ionized within detergent micelles or other membrane mimetics (24–27), which can then be removed from the membrane protein ions within the instrument. This method has been used to elucidate oligomeric state (28–30), complex organization (31, 32), and lipid interactions (33–35) of diverse membrane proteins. The addition of ion-mobility separation–mass spectrometry (IM-MS) provides data on the orientationally averaged size of analytes (36) and enables collision induced unfolding (CIU) experiments (37). In CIU, the energies experienced by gas-phase protein ions are increased in a stepwise fashion causing gas-phase protein unfolding to occur. These dynamic measurements have been shown to be sensitive to ligand binding (38, 39), glycosylation (40, 41), and disulfide bonding (40) in soluble proteins, as well as selective lipid and small molecule binding in membrane proteins (42–45). While CIU can clearly capture subtle structural changes in membrane proteins (43, 45, 46) and soluble mutant protein variants (47, 48) its ability to characterize membrane protein variants is only beginning to be explored.Here, we demonstrate the ability of native MS and CIU to detect key differences in the gas-phase stability and homodimer complex formation of PMP22 variants, together leading to insights into the mechanism of PMP22 dysregulation in disease. We quantify the propensity of PMP22 to dimerize across WT and seven disease-associated point mutations. We find that mutations associated with severe disease states form significantly more dimer than WT. Through CIU, we quantify the stability of gas-phase monomeric and dimeric PMP22 and find that variants bearing mutations associated with severe neuropathy exhibit the lowest relative monomer conformational stability. Interestingly, we also observe that dimers formed by various disease mutant forms of PMP22 are all more stable than WT PMP22 dimeric complexes. We continue by comparing our results to previously published biophysical datasets and find that our monomeric PMP22 gas-phase stability values correlate well with cellular trafficking data (6). Finally, we probe the effects of solubilization agents on PMP22 by characterizing its dimerization within sphingomyelin and cholesterol rich (SCOR) bicelles (49). We find that dimeric PMP22 complexes persist within SCOR bicelles and that the mutants resulting in the most severe disease phenotypes form higher population of dimer than WT. We conclude by describing a possible mechanism of PMP22 dysregulation in severe neurodegenerative diseases by which PMP22 monomers are destabilized, leading to dimers that traffic much less efficiently to the plasma membrane than WT PMP22.     

Structural insights into α-synuclein monomer–fibril interactions

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson’s disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

Synucleinopathies, including Parkinson’s disease (PD), are associated with the accumulation of intracellular neuronal aggregates termed as Lewy bodies and Lewy neuritis, which contain high concentration of the protein α-synuclein (α-Syn) in an aggregated state (1, 2). The disease-relevant role of α-Syn is further highlighted by mutations in the α-Syn gene (SNCA) causing familial PD [i.e., A30P (3), E46K (4), H50Q (5), G51D (6), A53E (7), and A53T (8)] and the duplication or triplication of the SNCA leading to early-onset PD in affected families (9, 10). α-Syn is a 140-residue intrinsically disordered protein (IDP) in solution (11) but adopts a helical structure in the presence of acidic lipid surfaces (12, 13). The positively charged N terminus (residues 1 to 60) is rich in lysine residues and contains KTKEGV binding repeats associated with vesicle binding (14). Moreover, the N-terminal domain includes all known SNCA familial PD mutations. The central region (residues 61 to 95) defines the non-amyloid-β component (NAC) (15), which is essential for α-Syn aggregation (16), while the C terminus (residues 96 to 140) is highly negatively charged.In vitro, α-Syn forms polymorphic amyloid fibrils (17–19) with unique arrangements of cross-β-sheet motifs (20–22). When injected into model animals, these fibrils induce a PD-like pathology (23) where the aggregation pathway of α-Syn plays a key role in the development of the disease (24). A detailed analysis of the aggregation kinetics of α-Syn into amyloids is therefore important toward understanding the toxic mechanisms relevant for synucleinopathies.Amyloid formation of α-Syn is very sensitive to solution conditions, including pH (25), temperature (26), and salt concentration (27). It further requires the presence of an air–water interface (28) or negatively charged lipid membranes (29) for which α-Syn has a high affinity. Previous studies suggest that amyloid fibril growth of α-Syn occurs via a nucleation-dependent polymerization reaction (30). Following a fairly slow primary nucleus formation, α-Syn fibrils are elongated by addition of single monomers. In a next step, the amyloid fibrils multiply by fragmentation or can catalyze the formation of new amyloids from monomers on their surface—a process known as secondary nucleation that was first described for sickle cell anemia 40 y ago (31). Fragmentation and secondary nucleation critically depend on the fibril mass and accelerate the aggregation kinetics (30). In the case of α-Syn aggregation under quiescent condition fragmentation does not exist and only the described secondary nucleation process occurs. While detailed kinetic experiments showed no significant secondary nucleation at pH 7, it strongly contributes at pH values lower than 6 (25, 30). However, mechanistic or structural information of the secondary nucleation process in α-Syn aggregation has been lacking so far.In this study we investigated the structural properties of α-Syn monomer–fibril interactions by NMR and electron paramagnetic resonance (EPR) spectroscopy. Our results provide insights into how monomeric α-Syn transiently interacts in vitro via its positively charged N terminus with the negatively charged C-terminal residues of the α-Syn fibrils, giving detailed insights into the mechanism of the secondary nucleation process.     

Segregation,integration, and balance of large-scale resting brain networks configure different cognitive abilities

Diverse cognitive processes set different demands on locally segregated and globally integrated brain activity. However, it remains an open question how resting brains configure their functional organization to balance the demands on network segregation and integration to best serve cognition. Here we use an eigenmode-based approach to identify hierarchical modules in functional brain networks and quantify the functional balance between network segregation and integration. In a large sample of healthy young adults (n = 991), we combine the whole-brain resting state functional magnetic resonance imaging (fMRI) data with a mean-filed model on the structural network derived from diffusion tensor imaging and demonstrate that resting brain networks are on average close to a balanced state. This state allows for a balanced time dwelling at segregated and integrated configurations and highly flexible switching between them. Furthermore, we employ structural equation modeling to estimate general and domain-specific cognitive phenotypes from nine tasks and demonstrate that network segregation, integration, and their balance in resting brains predict individual differences in diverse cognitive phenotypes. More specifically, stronger integration is associated with better general cognitive ability, stronger segregation fosters crystallized intelligence and processing speed, and an individual’s tendency toward balance supports better memory. Our findings provide a comprehensive and deep understanding of the brain’s functioning principles in supporting diverse functional demands and cognitive abilities and advance modern network neuroscience theories of human cognition.

The brain dynamically reconfigures its functional organization to support diverse cognitive task performances (1, 2). Successful reconfiguration underlying better task performance relies not only on sufficiently independent processing in specialized subsystems (i.e., segregation) but also on effective global cooperation between different subsystems (i.e., integration) (1–6). It has been observed that diverse cognitive tasks set different demands on segregation and integration (3, 5, 7–12). Higher segregation has been linked to simple motor tasks, and higher integration seems to underlie performance on tasks with a heavy cognitive load (8–12). However, it remains a great challenge to understand how the brain’s functional organization is configured to support heterogeneous demands on segregation and integration for diverse cognitive processes.Independently from specific task demands, the brain’s functional organization at rest can mirror relevant task-induced activity patterns and thus predict task performance (13, 14). Emerging evidence suggests that smaller differences between functional patterns at rest versus task states can facilitate better cognitive performance (13–16). Since diverse cognitive tasks differently demand on segregation and integration (3, 5, 7–12), the brain’s functional organization at rest is expected to possess the intrinsic capability of supporting diverse cognitive processes. Furthermore, previous studies suggest that healthy resting brains operate near a critical state to render the capability of rapidly exploring and switching in the brain’s state space with large operating repertoires (3, 13, 17–19). Resting brains are thus supposed to balance the segregation and integration (17, 20), so as to satisfy competing cognitive demands. However, this theory still lacks empirical evidence regarding whether large-scale brain networks at rest entail a balance between segregation and integration and whether the functional balance is associated with individual differences in cognitive abilities.To date, most of the relationships between brain functional configurations at rest and cognitive abilities are based on single tasks (7–12) that assess only specific aspects of cognition. General and domain-specific cognitive abilities are modeled at the latent level based on multiple tasks in differential psychology (21–24). These latent cognitive abilities are generalizations across tasks of the same domain and account for measurement error (25), and thus, they are much more suitable to reveal the neural basis of individual differences in human cognition. Recently, a cross-disciplinary network neuroscience theory (NNT) proposed a general framework to investigate the neural basis of cognitive abilities relying on system-wide topology characteristics and the dynamics of brain networks (26). According to NNT, brain networks functioning in an easy-to-reach state serve crystallized intelligence, whereas a difficult-to-reach state is needed for fluid intelligence (26). General cognitive ability, which is a statistical summary of fluid and crystallized intelligence, is considered to be facilitated by the capacity to flexibly switch between the above mentioned network states, i.e., an optimal balance between local and global processing (26). These predictions are still built upon a fragile empirical basis (27). Elucidating the relationship between functional balance and different cognitive abilities is crucial for validating and reframing NNT, particularly regarding the question of whether a functional balance in the brain at the resting state is beneficial for general cognitive ability of individuals.Before these questions can be robustly answered, the balance between segregation and integration in the large-scale brain must be explicitly defined and quantified. The modular structure of brain’s functional connectivity (FC) networks is known to provide the basis for specialized information processing within modules and the integration between them (8, 10, 15, 26, 28, 29). Although many studies have applied measures based on modules at a single level to study functional segregation and integration (7, 8, 10, 15, 28, 30), empirical evidence for a balance in the brain’s functional organization is still lacking. In fact, brain FC networks are hierarchically organized (31–33). Such an organization potentially supports nested segregation and integration across multiple levels. However, the classically applied modular partition at a single level does not allow the detection of hierarchical modules across multiple levels (34, 35). This insufficiency seems to be the main reason for the lack of a robust quantitative definition of the balance between segregation and integration.Here we explicitly identified the functional balance based on hierarchical modules of resting brain FC networks and explored associations with diverse cognitive abilities in a sample of 991 heathy young adults from the Washington University–University of Minnesota Consortium (WU-Minn) Human Connectome Project (HCP) (36). Using our previously published method called nested-spectral partition (NSP) based on eigenmodes (19), we first detected hierarchical modules in FC networks to propose an explicit balance measure. Second, we combined real data and a Gaussian linear model to demonstrate the functional balance in the group-averaged brain at the resting state. Then, we investigated individual differences in the balance and relationships to the temporal switching between segregated and integrated states. Finally, we applied structural equation modeling (SEM) to estimate latent factors of general and domain-specific cognitive abilities and investigated how segregation, integration, and their balance configure them.     

LGI1–ADAM22–MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1–ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv₁ channels, and LRRTM4–Neurexin adhesion molecules. Adam22^ΔC5/ΔC5 knock-in mice devoid of the ADAM22–MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1–ADAM22–MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Epilepsy, characterized by unprovoked, recurrent seizures, affects 1 to 2% of the population worldwide. Many genes that cause inherited epilepsy when mutated encode ion channels, and dysregulated synaptic transmission often causes epilepsy (1, 2). Although antiepileptic drugs have mainly targeted ion channels, they are not always effective and have adverse effects. It is therefore important to clarify the detailed processes for synaptic transmission and how they are affected in epilepsy.Recent superresolution imaging of the synapse reveals previously overlooked subsynaptic nano-organizations and pre- and postsynaptic nanodomains (3–6), and mathematical simulation suggests their nanometer-scale coordination in individual synapses for efficient synaptic transmission: presynaptic neurotransmitter release machinery and postsynaptic receptors precisely align across the synaptic cleft to make “transsynaptic nanocolumns” (7, 8).So far, numerous transsynaptic cell-adhesion molecules have been identified (9–12), including presynaptic Neurexins and type IIa receptor protein tyrosine phosphatases (PTPδ, PTPσ, and LAR) and postsynaptic Neuroligins, LRRTMs, NGL-3, IL1RAPL1, Slitrks, and SALMs. Neurexins–Neuroligins have attracted particular attention because of their synaptogenic activities when overexpressed and their genetic association with neuropsychiatric disorders (e.g., autism). Another type of transsynaptic adhesion complex mediated by synaptically secreted Cblns (e.g., Neurexin–Cbln1–GluD2) promotes synapse formation and maintenance (13–15). Genetic studies in Caenorhabditis elegans show that secreted Ce-Punctin, the ortholog of the mammalian ADAMTS-like family, specifies cholinergic versus GABAergic identity of postsynaptic domains and functions as an extracellular synaptic organizer (16). However, the molecular identity and in vivo physiological significance of transsynaptic nanocolumns remain incompletely understood.LGI1, a neuronal secreted protein, and its receptor ADAM22 have recently emerged as major determinants of brain excitability (17) as 1) mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (18); 2) mutations in the ADAM22 gene cause infantile epileptic encephalopathy with intractable seizures and intellectual disability (19, 20); 3) Lgi1 or Adam22 knockout mice display lethal epilepsy (21–24); and 4) autoantibodies against LGI1 cause limbic encephalitis characterized by seizures and amnesia (25–28). Functionally, LGI1–ADAM22 regulates AMPA receptor (AMPAR) and NMDA receptor (NMDAR)-mediated synaptic transmission (17, 22, 29) and Kv₁ channel-mediated neuronal excitability (30, 31). Recent structural analysis shows that LGI1 and ADAM22 form a 2:2 heterotetrameric assembly (ADAM22–LGI1–LGI1–ADAM22) (32), suggesting the transsynaptic configuration.In this study, we identify ADAM22-mediated synaptic protein networks in the brain, including pre- and postsynaptic MAGUKs and their functional bindings to transmembrane proteins (NMDA/AMPA glutamate receptors, voltage-dependent ion channels, cell-adhesion molecules, and vesicle-fusion machinery). ADAM22 knock-in mice lacking the MAGUK-binding motif show lethal epilepsy of hippocampal origin. In this mouse, postsynaptic PSD-95 nano-assembly as well as nano-scale alignment between pre- and postsynaptic proteins are significantly impaired. Importantly, PSD-95 is no longer able to modulate AMPAR-mediated synaptic transmission without binding to ADAM22. These findings establish that LGI1–ADAM22 instructs MAGUKs to organize transsynaptic nanocolumns and guarantee the stable brain activity.     

The intracellular environment affects protein–protein interactions

Protein–protein interactions are essential for life but rarely thermodynamically quantified in living cells. In vitro efforts show that protein complex stability is modulated by high concentrations of cosolutes, including synthetic polymers, proteins, and cell lysates via a combination of hard-core repulsions and chemical interactions. We quantified the stability of a model protein complex, the A34F GB1 homodimer, in buffer, Escherichia coli cells and Xenopus laevis oocytes. The complex is more stable in cells than in buffer and more stable in oocytes than E. coli. Studies of several variants show that increasing the negative charge on the homodimer surface increases stability in cells. These data, taken together with the fact that oocytes are less crowded than E. coli cells, lead to the conclusion that chemical interactions are more important than hard-core repulsions under physiological conditions, a conclusion also gleaned from studies of protein stability in cells. Our studies have implications for understanding how promiscuous—and specific—interactions coherently evolve for a protein to properly function in the crowded cellular environment.

Proteins rarely work alone. Networks of protein–protein interactions turn a myriad of chemical signals into physiological responses that maintain intracellular homeostasis (1). Therefore, it is unsurprising that nearly two-thirds of disease-causing missense mutations involve protein complexes (2). However, nearly all equilibrium thermodynamic and kinetic studies of protein–protein interactions have been performed in dilute buffer. Acquiring quantitative equilibrium data on protein complexes in cells, despite the fact that living things are not at equilibrium, is important for two reasons. First, the equilibrium condition is the starting point for estimating the driving force under nonequilibrium conditions (3). Second, recent results on protein stability show that conclusions from experiments conducted in dilute solution cannot be extrapolated to the crowded and complex intracellular environment (4).The cellular interior contains a variety of proteins, nucleic acids, and small molecules. In the bacterium Escherichia coli, the concentration of macromolecules can exceed 300 g/L and occupy up to 30% of volume (5). The macromolecule concentration in eukaryotic cells is smaller (6) but still hundreds of times larger than the solute concentrations used in most in vitro studies. Furthermore, the majority of proteins in the cells studied here, E. coli and oocytes, are net polyanions with mean isoelectric points of 6.6 and 6.7, respectively (7). Understanding how the intracellular environment modulates protein—and protein complex—stability is important because the totality of weak interactions in cells form the so-called quinary structure that organizes the crowded cellular interior (8–10). Importantly, these interactions cannot be measured in dilute solutions.The physical consequences of macromolecular crowding arise from two components: hard-core repulsions and so-called “soft” chemical interactions (11). Hard-core repulsion is a steric effect, arising from the impenetrable nature of atoms. Steric repulsions reduce the volume available to reactants and products. Simple theory predicts that sterics favor compact states (12). For small, folded proteins, stability is described by the two-state equilibrium between the biologically active folded state and the inactive, higher-energy, and less-compact unfolded state (13). For protein complex formation involving folded proteins, which comprises an equilibrium between constituent proteins and the complex, the complex exposes less surface than the sum of the two monomers (14). Thus, simple theory predicts stabilization of proteins and protein complexes, although more sophisticated theoretical efforts focused on the size of crowding molecule predict stabilization or destabilization (15, 16).Soft interactions include hydrogen bonds and charge–charge, water–protein, solute–protein, and hydrophobic interactions. Of these, the only strong repulsive interaction is that from the opposition of like charges. These repulsions add to the hard-core effect and are therefore stabilizing. The other soft interactions are attractive and destabilizing because they favor expanded conformations that allow access to the attractive surfaces.Until recently the focus was on hard-core interactions and crowding (17) because studies of protein stability (quantified as the free energy of unfolding, ΔGUo'') in uncharged synthetic polymers tended to show only a stabilizing influence compared to buffer alone (11). However, recent studies of stability under more physiologically relevant crowded conditions and in living cells show, by and large, a decrease in stability (4). Studies of protein–protein interactions in vitro under physiologically relevant crowded conditions also show the importance of chemical interactions (18, 19).Yeast two-hybrid techniques, coimmunoprecipitation, and split systems provide simple yes/no information about protein interactions in cells, however, these methodologies are also prone to false positives. The protein–protein interface can also be characterized by in-cell NMR. However, quantification of the equilibrium thermodynamics of complex stability is challenging in cells. The few reports tend to involve heterodimerization (20), which can be complicated to assess because the concentration of both reactants must be controlled, fluorescence-resonance-energy transfer because labels adding hundreds to thousands of daltons must be included, or because competition with natural, unlabeled proteins in cells must be considered (21–27).     

Homocysteine fibrillar assemblies display cross-talk with Alzheimer’s disease β-amyloid polypeptide

High levels of homocysteine are reported as a risk factor for Alzheimer’s disease (AD). Correspondingly, inborn hyperhomocysteinemia is associated with an increased predisposition to the development of dementia in later stages of life. Yet, the mechanistic link between homocysteine accumulation and the pathological neurodegenerative processes is still elusive. Furthermore, despite the clear association between protein aggregation and AD, attempts to develop therapy that specifically targets this process have not been successful. It is envisioned that the failure in the development of efficacious therapeutic intervention may lie in the metabolomic state of affected individuals. We recently demonstrated the ability of metabolites to self-assemble and cross-seed the aggregation of pathological proteins, suggesting a role for metabolite structures in the initiation of neurodegenerative diseases. Here, we provide a report of homocysteine crystal structure and self-assembly into amyloid-like toxic fibrils, their inhibition by polyphenols, and their ability to seed the aggregation of the AD-associated β-amyloid polypeptide. A yeast model of hyperhomocysteinemia indicates a toxic effect, correlated with increased intracellular amyloid staining that could be rescued by polyphenol treatment. Analysis of AD mouse model brain sections indicates the presence of homocysteine assemblies and the interplay between β-amyloid and homocysteine. This work implies a molecular basis for the association between homocysteine accumulation and AD pathology, potentially leading to a paradigm shift in the understanding of AD initial pathological processes.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, currently affecting tens of millions of individuals globally and leading to immense social and financial impacts (1, 2). Despite the vast efforts to develop a pharmacological treatment for the disease, recent clinical trials had failed (3, 4). While it is very clear that amyloid formation is associated with AD (5, 6) and genetic variations in the gene coding for the β-amyloid polypeptide are associated with early onset of the disease (7), direct targeting of the aggregation of AD-associated pathological proteins and polypeptides has so far not resulted in clinical development of any disease-modifying treatment (8–10). Therefore, there is an essential need to understand the early biological changes that may induce the diverse pathologies observed in AD and other forms of dementia in order to identify new therapeutic targets (10).In the last decades, an enormous body of research had provided important information on the amyloid cascade, oxidative stress, and inflammatory response that are involved in AD (3, 11). Specifically, the notion of β-amyloid toxicity and extracellular plaque formation as the main cause of neuronal and synaptic loss has been extensively studied (12–14). Yet, the key to AD treatment and prevention remains elusive, and the initial steps leading to the formation and accumulation of these protein aggregates and the role of nonproteinaceous agents in this process are still not understood (10). Since only 1% of AD cases result from a familial mutation and the majority of patients are sporadic (2, 4), it is important to explore new directions to understand the biological mechanisms underlying these cases. Studies of metabolite profiling in brain regions and body fluids of AD and Parkinson’s disease (PD) patients show interference with specific metabolic pathways (15–19). Homocysteine (Hcy), a noncoded amino acid, was identified as a major risk factor for AD as high plasma concentrations were associated with the progression of the disease (20–22). Higher Hcy serum concentration was also correlated with behavioral and psychological symptoms of AD (23) and was associated with changes in motor function and cognitive decline in PD as well as with a more severe cognitive impairment in elderly adults (24–26). Furthermore, it was shown that significantly decreased hippocampal and cortical volume is associated with increased Hcy plasma concentration (27).Cystathionine β-synthase (CBS) deficiency, leading to excess of Hcy, results in the hyperhomocysteinemia inborn error of metabolism (IEM) disorder characterized by severe cognitive consequences (28–30). While elevated plasma Hcy is frequently reported as a strong and independent risk factor for the development of cognitive decline and dementia, the mechanism of its involvement is elusive (20, 31–34). Interestingly, the association between Hcy and AD-related pathological proteins was also demonstrated. Hcy-rich medium was shown to be cytotoxic to hippocampal and cortical neurons, resulting in increased β-amyloid–induced cell death (35–37). In addition, Hcy was found to bind β-amyloid_1-40, thereby stimulating β-sheet structure formation to facilitate its deposition. Indeed, induced Hcy accumulation in the brains of rats caused an elevation of β-amyloid deposition (38, 39). Furthermore, Hcy increased total tau and phosphorylated tau protein levels as well as the level of tau oligomers (40). Although the involvement of Hcy accumulation in AD pathology is evidential, no mechanistic insight has so far been suggested.We have previously demonstrated that small metabolites can self-assemble into amyloid-like structures with amyloidogenic characteristics (41–44). The presence of metabolite assemblies in IEM disorders (e.g., phenylalanine in Phenylketonuria) exemplifies their physiological importance in pathologically diversified diseases. Interestingly, recent studies demonstrated that metabolite assemblies could cross-seed the aggregation of proteins under physiological conditions, thereby suggesting a possible mechanism in which accumulated metabolites interfere with protein function and folding (45, 46). Assemblies of quinolinic acid, an endogenous neurometabolite that is involved in the pathology of PD, induce the aggregation of α-synuclein both in vitro and in cell culture (45). In addition, phenylalanine preformed fibrils were shown to initiate the aggregation of several proteins under physiological conditions (46). Metabolite accumulation, structure formation, and seeding of proteins may thus underlie the unknown role of metabolites in neurodegenerative pathologies (47). Specifically, seeding of amyloidogenic proteins by preformed fibrils may be part of the mechanisms underlying the stereotypical spreading of toxic aggregates in the brains of AD patients.We recently established an in vivo yeast model for IEM disorders by genetically modifying the yeast to reflect the mutations found in patients showing accumulation of the adenine nucleobase and its derivatives (48). Yeast model systems provide a powerful platform to elucidate the pathophysiology of diseases, as well as for the screening and development of disease-modifying therapeutics (49). This model was found to be a valid system, as supported by its robust sensitivity to adenine feeding and by the fact that adenine supramolecular structures could be detected. Furthermore, the addition of a generic fibrillation-modifying polyphenolic compound rescued the toxic effect without lowering the concentration of adenine, indicating the therapeutic potential of our model for the modulation of structure formation (48, 50).Here, aiming to explore the role of Hcy in AD, we demonstrate the formation of amyloid-like fibrils by Hcy in vitro and in vivo in a yeast model. Structural characterization of the Hcy fibrils and their cytotoxic effect were both studied. In addition, we revealed that polyphenolic inhibitors could rescue the toxic effect of Hcy assemblies and inhibit its structure formation. Remarkably, immunohistochemistry allowed the detection of Hcy fibrils in the brain of AD model mice as well as the apparent interplay between Hcy and β-amyloid. Finally, we demonstrated the cross-seeding of AD-related pathological protein by Hcy assemblies. Our work suggests a research direction for the association between metabolite accumulation and the initiation of neurodegenerative processes, thus offering a path for the development of therapeutic treatments that will target the key early stages of the disease.     

Tau forms oligomeric complexes on microtubules that are distinct from tau aggregates

Tau is a microtubule-associated protein, which promotes neuronal microtubule assembly and stability. Accumulation of tau into insoluble aggregates known as neurofibrillary tangles (NFTs) is a pathological hallmark of several neurodegenerative diseases. The current hypothesis is that small, soluble oligomeric tau species preceding NFT formation cause toxicity. However, thus far, visualizing the spatial distribution of tau monomers and oligomers inside cells under physiological or pathological conditions has not been possible. Here, using single-molecule localization microscopy, we show that tau forms small oligomers on microtubules ex vivo. These oligomers are distinct from those found in cells exhibiting tau aggregation and could be precursors of aggregated tau in pathology. Furthermore, using an unsupervised shape classification algorithm that we developed, we show that different tau phosphorylation states are associated with distinct tau aggregate species. Our work elucidates tau’s nanoscale composition under nonaggregated and aggregated conditions ex vivo.

Tau is a microtubule-associated protein mainly expressed in neurons. Tau is an intrinsically disordered protein, which facilitates the assembly and stability of neuronal microtubules. Immunofluorescence labeling showed that tau is highly abundant in neuronal axons and shows a graded distribution with higher concentration toward the axon terminal (1). Tau binding is thought to stabilize neuronal microtubules, although this idea has recently been called into question, and recent work suggests that tau may bind the more labile part of microtubules, enabling them to have long labile domains (2). The first near-atomic model of tau bound to microtubules obtained using cryogenic electron microscopy (cryo-EM) and computational modeling revealed that tau binds along the microtubule protofilament, stabilizing the interface between tubulin dimers (3). Tau has also been shown to undergo liquid–liquid phase separation in vitro, and tau condensates have been proposed to facilitate microtubule polymerization due to their ability to sequester soluble tubulin (4). In vitro reconstitution experiments further showed that tau forms liquid condensates that are several microns in size on taxol-stabilized microtubules, and these tau “islands” regulate microtubule severing and microtubule–motor protein interactions (5, 6). However, despite progress, the distribution of tau on microtubules in intact cells and the physiological relevance of tau condensates are unclear.Under pathological conditions, tau becomes hyperphosphorylated and detaches from microtubules, leading to the misfolding and formation of tau aggregates in the cytosol (7). Accumulation of tau into insoluble aggregates known as neurofibrillary tangles (NFTs) is a hallmark of several neurodegenerative diseases such as Alzheimer’s disease (AD) and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), which are collectively known as tauopathies. Liquid phase separation has also been suggested to play a role in pathological tau aggregation, as aggregation-prone tau mutants have an increased propensity to undergo liquid phase separation (4). Recently, cryo-EM studies have also revealed the high-resolution structures of insoluble tau aggregates, including paired helical filaments (PHF) and straight filaments (SFs) from AD brain (8). While these insoluble aggregates such as PHFs, SFs, and NFTs have traditionally been considered the pathological tau species, recent evidence from animal models has shown that neurodegeneration in terms of synaptic dysfunction and behavioral abnormalities can exist without the presence of NFTs (9, 10). The current hypothesis is that earlier stages of tau aggregation initiate pathology. In particular, small, soluble tau oligomeric species preceding the formation of NFTs could be the cause of loss of tau function and toxicity. Soluble tau oligomers have been isolated from homogenized AD brains and detected on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels (11). These tau oligomers have also been implicated in synaptic loss in transgenic mice expressing wild-type human tau (12–14). Moreover, injection of tau oligomers rather than monomers or fibrils into the brain of wild-type mice was sufficient to produce cognitive and synaptic abnormalities (15, 16). Tau oligomers can lengthen and adopt a β-sheet conformation, which renders them detergent insoluble. Such oligomers have granular appearance under atomic force microscopy (AFM) (17). Despite their in vitro characterization, to date, there have not been studies visualizing and characterizing the formation of these small oligomers in intact cells due to the lack of quantitative, high-resolution tools. The lack of sensitive tools that enable visualizing the spatial distribution and quantifying the stoichiometry of tau complexes within native and diseased neurons prevents progress in studying the mechanisms that lead to pathological tau oligomerization and the impact of early stages of tau aggregation on cellular processes.Here, we have used quantitative super-resolution microscopy to visualize and determine the nanoscale organization of tau in various engineered cells modeling the nonaggregated state of tau (QBI-293 Clone 4.0 cells expressing 4R-P301L-GFP tau, BSC-1 cells expressing 3R-WT-GFP tau, and BSC-1 cells expressing 4R-WT-GFP tau) (18) as well as in rat hippocampal neurons. In addition, using an engineered cell model expressing tau harboring the FTDP-17 mutation P301L and transduced with exogenous tau fibrils to mimic tau aggregation in disease (QBI-293 Clone 4.1 cells expressing 4R-P301L-GFP tau) (18), we characterized the nature of tau aggregates including oligomeric tau species. Surprisingly, our results show that, in engineered cell lines modeling a nonaggregated state of tau, tau forms a patchy distribution consisting of nanoclusters along the microtubule. Tau nanoclusters mainly correspond to monomeric, dimeric, and trimeric tau complexes. The nanoscale distribution of microtubule-associated tau in these engineered cells resembles the nanoscale distribution of microtubule-associated endogenous tau in hippocampal neurons isolated from rats. In engineered cell lines modeling an aggregated state of tau (QBI-293 Clone 4.1), tau forms a diverse range of aggregate species, including small oligomeric tau assemblies, small fibrillary structures, branched fibrils, and large plaque-like structures resembling NFTs. The oligomeric species found in the aggregated tau cell model are partially microtubule associated and are distinct from the monomers, dimers, and trimers found in the nonaggregated cell models and in neurons, since they are larger, containing more tau molecules. Using antibodies that recognize different phosphorylation states of tau and an unsupervised machine learning–based shape classification method, we further show that different phosphorylation states associate with distinct higher-order tau aggregate species. Overall, we present a detailed, quantitative characterization of tau oligomers on microtubules in intact cells and of tau aggregates with nanoscale resolution in a cell model of FTDP-17.     

Periodic training of creeping solids

We consider disordered solids in which the microscopic elements can deform plastically in response to stresses on them. We show that by driving the system periodically, this plasticity can be exploited to train in desired elastic properties, both in the global moduli and in local “allosteric” interactions. Periodic driving can couple an applied “source” strain to a “target” strain over a path in the energy landscape. This coupling allows control of the system’s response, even at large strains well into the nonlinear regime, where it can be difficult to achieve control simply by design.

Metamaterials offer the possibility of creating a broad array of behaviors not found in ordinary, nonarchitected materials. By manipulating the connectivity and strength of the structural units, rather than the composition or structure of the native material itself, metamaterials with unusual elastic response can be designed. These include systems that display phononic band gaps (1, 2), negative Poisson’s ratios (3–7), negative-compressibility transitions (8), topologically protected modes (9, 10), negative swelling (11), complex pattern formation (12), and allostery-inspired responses, where the imposed strain on a given site is propagated to a distant target (13–15). While such metamaterials can be designed and built on a relatively small scale, it is not always possible to scale up the number of components or to control the microstructure at the microscopic level in order to achieve the desired behavior—especially when the applied deformations are well outside the linear-response regime.One successful approach has been to base the design of metamaterials on a repeating unit cell. Such an approach requires precise control of each degree of freedom during fabrication of the unit cell, but has the advantage that each unit cell is the same. However, this strategy cannot create a material with a heterogeneous or localized response, which, by definition, cannot be captured by identical responses in each unit cell. A mechanical metamaterial with inhomogeneous response is difficult to design, since it requires detailed knowledge of structure and mechanics at the constituent scale and computer resources that grow with system size. It is also challenging to fabricate, since it requires control and manipulation at the constituent scale.The idea of “directed aging” (16) circumvents these obstacles by starting with a disordered solid and training it while it ages by applying appropriate stresses in such a way that it ultimately evolves to have the desired functionality. Directed aging takes advantage of the natural tendency of a material to minimize its energy under stress by deforming plastically. A demonstrated process that can be understood in terms of directed aging is the heating of solid foams under pressure to create auxetic (i.e., negative Poisson’s ratio) foams (3). The concept of directed aging allows the creation of materials with a variety of responses determined by the stresses applied (16). Aging a system under a fixed shear strain (as opposed to compressive strain), for example, leads to systems with high Poisson’s ratios. The challenge for this general approach is to find appropriate flexible protocols for the training that will produce a broad class of response.In this paper, we explore the effects of applying a periodic training strain instead of a fixed training strain. The evolution of materials under periodic strains has been extensively studied in contexts such as strain hardening (17–19) and material fatigue (20–22). Here, we develop a potentially generalizable strategy for how to design periodic strains to obtain desired complex responses in systems that deform plastically and irreversibly via creep. To demonstrate this approach, we start with the model systems and train initially identical systems to exhibit three different types of responses: negative Poisson’s ratio, bistability, and mechanical allosteric response (13–15). We find that we can control responses far into the nonlinear regime.     

Cell type-specific lipid storage changes in Parkinson’s disease patient brains are recapitulated by experimental glycolipid disturbance

Neurons are dependent on proper trafficking of lipids to neighboring glia for lipid exchange and disposal of potentially lipotoxic metabolites, producing distinct lipid distribution profiles among various cell types of the central nervous system. Little is known of the cellular distribution of neutral lipids in the substantia nigra (SN) of Parkinson’s disease (PD) patients and its relationship to inflammatory signaling. This study aimed to determine human PD SN neutral lipid content and distribution in dopaminergic neurons, astrocytes, and microglia relative to age-matched healthy subject controls. The results show that while total neutral lipid content was unchanged relative to age-matched controls, the levels of whole SN triglycerides were correlated with inflammation-attenuating glycoprotein non-metastatic melanoma protein B (GPNMB) signaling in human PD SN. Histological localization of neutral lipids using a fluorescent probe (BODIPY) revealed that dopaminergic neurons and midbrain microglia significantly accumulated intracellular lipids in PD SN, while adjacent astrocytes had a reduced lipid load overall. This pattern was recapitulated by experimental in vivo inhibition of glucocerebrosidase activity in mice. Agents or therapies that restore lipid homeostasis among neurons, astrocytes, and microglia could potentially correct PD pathogenesis and disease progression.

Both neurons and glia depend on tight regulation and exchange of lipids for proper function. Typically through lipid transport mechanisms (1, 2), the continuous exchange of lipids is essential for maintaining physiological function as brain lipid content, transport, and distribution are complex and critical aspects of neuropathology. Recently, both clinical findings and experimental studies have implicated lipid storage and trafficking in the pathogenesis of Parkinson’s disease (PD) and related disorders (3, 4). Reduced function of lysosomal hydrolases that are associated with lysosomal storage disorders increases the risk for PD and results in brain pathology similar to that seen in most sporadic and genetic forms of the disease (5–11).One of the strongest genetic risk factors for PD is heterozygous loss-of-function mutations in GBA1, encoding the lysosomal hydrolase glucocerebrosidase (GCase) (12–14), a deficiency that causes systemic accumulation of its glycolipid substrate glucosylceramide (GlcCer). GCase activity is reduced with aging of both the human and murine brain (6, 15), and age is the overall greatest risk factor for developing PD (16). In contrast, the more severe loss of GCase activity in homozygous GBA1 mutant carriers causes the lysosomal storage disease Gaucher disease (GD) (17). Conduritol beta epoxide (CBE), an irreversible inhibitor of GCase, causes widespread accumulation of GlcCer and related glycosphingolipids in mice. In this model, there is marked increase of high molecular weight alpha-synuclein (aSYN) and deposition of proteinase K-resistant aSYN resembling that seen in PD (18–20). aSYN is a constituent of the classical Lewy bodies and Lewy neurites (21) found in surviving dopaminergic neurons in postmortem PD materials as a standard pathological criterion for PD (22). aSYN has a lipid-binding domain, and aSYN protein–lipid interactions are potentially perturbed in PD (reviewed in refs. 17, 23, 24). We recently demonstrated that excessive aSYN can deposit into lipid compartments, and that this process is reversible under increased lysosomal β-hexosaminidase expression (25).Several in vitro physiological studies have demonstrated that a reduction in neuronal neutral lipid storage or knockdown of fatty acid desaturases protects cultured neurons from degeneration (26–28). However, as neurons exhibit limited capacity to synthesize, metabolize, and transport lipid species under physiological conditions (29), other resident cells of the substantia nigra (SN), such as glial cells, are required to maintain lipid homeostasis in the brain. Astrocyte health and lipid exchange function—and microglial activation—are potentially central to PD pathogenesis and other age-dependent neurodegenerative diseases (30–33). Brain resident microglia can accumulate and generate lipids, which may propagate inflammatory processes through, for example, TREM2 binding of apolipoproteins (34–38). Proinflammatory cytokines present in chronic conditions are attenuated by the binding of glycoprotein nonmetastatic melanoma protein B (GPNMB) to the CD44 receptor on astrocytes (39). In GD patient serum, GPNMB level is significantly correlated with disease severity (40), and GPNMB is increased in human PD SN and following CBE-induced glycolipid accumulation in mice (41).There are surprisingly little data on lipid distribution patterns in PD-affected cell types given the relevance of lipid homeostasis, aSYN–lipid interactions, and lipidopathy-associated inflammatory signatures as they relate to PD (17, 41). In this study, we measured the differences in total lipid content and cellular distribution between PD and healthy subject (HS) SN and compared them with a lipid-associated neuroinflammatory signal, GPNMB. To quantify cell type-specific intracellular lipid content, colocalization analysis was performed on human postmortem SN sections that were costained for neutral lipids and markers of dopaminergic neurons, astrocytes, and microglia. Compared with HS SN, the lipid content of PD dopaminergic neurons and microglia was significantly higher, and that of astrocytes was significantly lower. To understand a possible mechanistic reason for this lipid distribution pattern, we used an in vivo mouse model of glycolipid dysregulation and attendant aSYN accumulation through GCase inhibition by CBE. This in vivo model recapitulated the lipid distribution pattern that we observed in human PD SN. Based on these data, we propose that PD is characterized by a unique neutral lipid distribution signature in neurons, astrocytes, and microglia that can be recapitulated experimentally by glycolipid dysregulation.     

Wild-type α-synuclein inherits the structure and exacerbated neuropathology of E46K mutant fibril strain by cross-seeding

Heterozygous point mutations of α-synuclein (α-syn) have been linked to the early onset and rapid progression of familial Parkinson’s diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) α-syn and its role in the exacerbated pathology of α-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant α-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different α-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryo-electron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT α-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT α-syn in such a way that the mutant pathology would be amplified in fPD.

α-Synuclein (α-Syn) is the main component of Lewy bodies, which serve as the common histological hallmark of Parkinson’s disease (PD) and other synucleinopathies (1, 2). α-Syn fibrillation and cell-to-cell transmission in the brain play essential roles in disease progression (3–5). Interestingly, WT α-syn could form fibrils with distinct polymorphs, which exhibit disparate seeding capability in vitro and induce distinct neuropathologies in mouse models (6–10). Therefore, it is proposed that α-syn fibril polymorphism may underlie clinicopathological variability of synucleinopathies (6, 9). In fPD, several single-point mutations of SNCA have been identified, which are linked to early-onset, severe, and highly heterogeneous clinical symptoms (11–13). These mutations have been reported to influence either the physiological or pathological function of α-syn (14). For instance, A30P weakens while E46K strengthens α-syn membrane binding affinity that may affect its function in synaptic vesicle trafficking (14, 15). E46K, A53T, G51D, and H50Q have been found to alter the aggregation kinetics of α-syn in different manners (15–17). Recently, several cryogenic electron microscopy (cryo-EM) studies revealed that α-syn with these mutations forms diverse fibril structures that are distinct from the WT α-syn fibrils (18–26). Whether and how hereditary mutations induced fibril polymorphism contributes to the early-onset and exacerbated pathology in fPD remains to be elucidated. More importantly, most fPD patients are heterozygous for SNCA mutations (12, 13, 27, 28), which leads to another critical question: could mutant fibrils cross-seed WT α-syn to orchestrate neuropathology in fPD patients?E46K mutation is one of the eight disease-causing mutations on SNCA originally identified from a Spanish family with autosomal-dominant PD (11). E46K-associated fPD features early-onset motor symptoms and rapid progression of dementia with Lewy bodies (11). Studies have shown that E46K mutant has higher neurotoxicity than WT α-syn in neurons and mouse models overexpressing α-syn (29–32). The underlying mechanism is debatable. Some reported that E46K promotes the formation of soluble species of α-syn without affecting the insoluble fraction (29, 30), while others suggested that E46K mutation may destabilize α-syn tetramer and induce aggregation (31, 32). Our previous study showed that E46K mutation disrupts the salt bridge between E46 and K80 in the WT fibril strain and rearranges α-syn into a different polymorph (33). Compared with the WT strain, the E46K fibril strain is prone to be fragmented due to its smaller and less stable fibril core (33). Intriguingly, the E46K strain exhibits higher seeding ability in vitro, suggesting that it might induce neuropathology different from the WT strain in vivo (33).In this study, we found that human E46K and WT fibril strains (referred to as hE46K and hWT strains) induced α-syn aggregates with distinct morphologies in mice. Mice injected with the hE46K strain developed more α-syn aggregation and early-onset motor deficits compared with the mice injected with the hWT strain. Notably, the hE46K strain was capable of cross-seeding both human and mouse WT (mWT) α-syn to form fibrils (named as hWT_cs and mWT_cs). The cross-seeded fibrils replicated the structure and seeding capability of the hE46K template both in vitro and in vivo. Our results suggest that the hE46K strain could propagate its structure as well as the seeding properties to the WT monomer so as to amplify the α-syn pathology in fPD.