==== Front J Clin Invest J Clin Invest J Clin Invest The Journal of Clinical Investigation 0021-9738 1558-8238 American Society for Clinical Investigation 168549 10.1172/JCI168549 Review Disrupting pathologic phase transitions in neurodegeneration Hurtle Bryan T. Hurtle.Bryan@medstudent.pitt.edu 1234 http://orcid.org/0009-0006-5395-5160 Xie Longxin XIEL@pitt.edu 345 http://orcid.org/0000-0002-2383-9015 Donnelly Christopher J. chrisdonnelly@pitt.edu 1234 1 Center for Neuroscience at the University of Pittsburgh Graduate Program; 2 Medical Scientist Training Program, University of Pittsburgh; and 3 LiveLikeLou Center for ALS Research at the University of Pittsburgh Brain Institute; Pittsburgh, Pennsylvania, USA. 4 Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. 5 School of Medicine, Tsinghua University, Beijing, China. Address correspondence to: Christopher J. Donnelly, Biomedical Science Tower 3, 3501 Fifth Avenue, 6th Floor, Room 6061, Pittsburgh, Pennsylvania 15261, USA. Email: cjdon25@pitt.edu. Authorship note: BTH and LX are co–first authors. 3 7 2023 3 7 2023 3 7 2023 133 13 e168549© 2023 Hurtle et al. 2023 Hurtle et al. https://creativecommons.org/licenses/by/4.0/ This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. This article is available online at https://www.jci.org/articles/view/168549 Solid-like protein deposits found in aged and diseased human brains have revealed a relationship between insoluble protein accumulations and the resulting deficits in neurologic function. Clinically diverse neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, frontotemporal lobar degeneration, and amyotrophic lateral sclerosis, exhibit unique and disease-specific biochemical protein signatures and abnormal protein depositions that often correlate with disease pathogenesis. Recent evidence indicates that many pathologic proteins assemble into liquid-like protein phases through the highly coordinated process of liquid-liquid phase separation. Over the last decade, biomolecular phase transitions have emerged as a fundamental mechanism of cellular organization. Liquid-like condensates organize functionally related biomolecules within the cell, and many neuropathology-associated proteins reside within these dynamic structures. Thus, examining biomolecular phase transitions enhances our understanding of the molecular mechanisms mediating toxicity across diverse neurodegenerative diseases. This Review explores the known mechanisms contributing to aberrant protein phase transitions in neurodegenerative diseases, focusing on tau and TDP-43 proteinopathies and outlining potential therapeutic strategies to regulate these pathologic events. ==== Body pmcIntroduction Neurodegenerative diseases (NDDs) are a heterogeneous class of incurable and debilitating disorders characterized by the progressive degeneration of vulnerable cell populations in the central nervous system (CNS). Decades of research investigating the most common NDDs, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal lobar degeneration (FTLD), and amyotrophic lateral sclerosis (ALS), revealed clinical and neuropathologic heterogeneity between, and within, these diseases (1–3). However, NDDs display a fundamental commonality — proteins soluble under physiologic conditions accumulate into solid-like pathologic protein inclusions, and this is associated with clinical progression (4, 5). Furthermore, disease-causing mutations in genes that encode proteins that pathologically accumulate, such as amyloid-β (APP) in AD, tau (MAPT) in FTLD-tau, TDP-43 (TARDBP) in ALS/FTLD–TDP-43, and α-synuclein (SNCA) in PD, cause familial forms of each disease (6–8). Sporadic-NDD patients with unclear familial inheritance and no genetic mutation in the genes that encode these proteins similarly present with neuropathologic deposits of the wild-type protein in the CNS. Furthermore, these sporadic NDDs often display remarkably similar clinical syndromes when compared with the familial form of the disease (2, 9). Multidisciplinary efforts have gone into understanding mechanisms through which tau and TDP-43 proteins regulate neuronal homeostasis and contribute to NDDs (10–13). These efforts revealed considerable clinical overlap between tau and TDP-43 proteinopathies (14–16). In addition to AD, the most common NDD, solid self-assemblies of tau are found in related dementias termed “tauopathies,” including FTLD-tau, corticobasal degeneration (CBD), Pick’s disease, progressive supranuclear palsy (PSP), and chronic traumatic encephalopathy (CTE) (17). While several mutations in the TARDBP gene contribute to a small percentage of ALS and FTLD–TDP-43 cases, mislocalized and insoluble TDP-43 self-assemblies are found in up to about 97% of individuals with sporadic ALS, up to about 85% with CTE, about 45% with FTLD, and about 40%–60% with AD (18, 19). Several recent studies characterized the ability of these proteins to undergo liquid-like phase separation under physiologic conditions, often into membraneless organelles (13, 20–24). Accordingly, the incidence of tau and TDP-43 pathology across genetic and sporadic NDDs likely highlights a convergence of several upstream mechanisms driving aberrant protein phase transitions and disease progression. In the following sections, we will explore the relationship between protein structure, biological phase transitions, protein self-assembly, and the organization of multicomponent condensates using tau and TDP-43 as representative proteins. Later sections will survey diverse targeting strategies proposed for tau and TDP-43 proteinopathies, focusing on how protein phase transitions and condensate assembly mechanisms can be leveraged as potential therapeutic avenues of intervention. Protein self-assembly through homotypic phase transitions Intracellular NDD-associated proteins self-assemble into diverse polymeric structures and liquid-like protein phases capable of organizing functionally related proteins, nucleic acids, and various biomolecules (13, 22, 25–27). Physiologically, most proteins are soluble and exist in a liquid-like state. In a simple system, e.g., a purified protein in solution, a protein will be soluble when the attractive interactions between different molecules are low enough to maintain a well-mixed state (26, 28, 29). Raising the protein concentration or modifying the balance of attractive and repulsive forces may exceed a protein’s saturation concentration (Csat) and precipitate a new and denser phase. In this context, where the protein is lacking complex biomolecular interactions, homotypic interactions regulate a segregative protein phase transition, which results in a new, denser phase (Cdense) coexisting within the dilute phase (28, 30, 31). Strong driving forces for a given phase transition are generated by lowering of the Csat, a context-dependent property affected by factors intrinsic to a protein’s sequence, localized concentration, cell size changes, temperature, pH, and ionic environments (28, 32–35). Phase transitions that give rise to two coexisting phases can be liquid-like or display solid-like properties. The appropriate prefix (liquid-like, solid-like) depends on the material properties of the emerging phase. Defining characteristics that distinguish liquid-like protein phases include rapid reversibility, interior molecular diffusion, and the ability to exchange molecules with the surrounding phase (i.e., cytosol) (26, 36). Notably, liquids may transition into solid-like states that emerge through several processes, including gelation, requiring networks of interactions (gel-like), or age-dependent increases in viscosity (glass-like). Solids can also emerge from liquid-like phases by forming fibrils or crystal-like aggregates such as amyloids. The human proteome is a continuum of protein structures ranging from intrinsically folded proteins to intrinsically disordered proteins (IDPs), with most containing both ordered domains and intrinsically disordered regions (IDRs) (28, 37–39). IDRs are regions of a protein sequence that lack well-defined secondary and tertiary structures. Phase separation and condensate-promoting features include modular interaction domains and stretches of low-complexity sequences found within IDRs. Tau and TDP-43 are modular, multivalent proteins with IDRs that enable and regulate homotypic and heterotypic interactions to generate complex and context-dependent molecular interactions (10, 23, 40–42). These protein architectures tune the concentration required for phase separation and dictate the resultant assembly and material states. Importantly, proteins with IDRs exist in a dynamic equilibrium of conformationally distinct states, and the structural properties of IDRs can quickly adjust owing to changes in solution conditions, posttranslational modifications (PTMs), or interactions with other molecules (26, 34, 36, 43–45). The generation of neurotoxic self-assemblies represents a fundamental transformation during the pathogenesis of NDDs (4, 5, 17, 46–48). In vitro experiments using purified proteins, work in transgenic animal disease models, and studies with postmortem human brain tissue show that both tau and TDP-43 self-assemble into polymeric states with varying structures and material properties (17, 49–59). Self-polymerization can occur under dilute conditions or within liquid-like droplets. However, pathogenic tau and TDP-43 self-assemblies rely on the exposure of small aggregation-prone regions, including steric zippers or low-complexity, aromatic-rich, kinked segments (LARKS) (60, 61). Disruption in protein conformation due to intrinsic factors (NDD-causing missense mutations, PTMs) or extrinsic factors (biomolecular interactions, cellular environment) may expose these buried short, aggregation-prone sequences in proteins (24, 62). Importantly, specific conformational transformations of protein monomers that are capable of nucleating stable self-interactions with other monomers are required (48, 63). These structural conformations drive unique assembly pathways specific to that protein, which ultimately translates to distinct pathologies observed in NDDs (64). Recent groundbreaking cryo–electron microscopy studies found that specific conformations underlie clinical subtypes of tau and TDP-43 pathologies. Remarkably, an increasing number of clinical presentations and neuropathologic findings correlate with structurally specific fibrils of varying biophysical properties and cellular effects (51–53, 56–58). While short aggregation-prone sequences necessary for pathologic self-assemblies have been discovered in tau and TDP-43 (steric zippers and LARKS), several other regions within these two proteins can regulate phase transition behavior. TDP-43 contains a C-terminal domain (CTD) that comprises two disordered regions (IDR1, IDR2) and a short α-helical fold (CR helix) that is stabilized by adjacent homomeric contacts between other TDP-43 CR helices (Figure 1A) (23, 65). This CTD, also defined as a low-complexity domain (LCD), is sufficient for liquid-like phase separation and likely important for physiologic functions, including RNA splicing (24, 66, 67). However, current experimental evidence supports a TDP-43 oligomerization model where physically distant regions regulate the ability to nucleate self-assembly. While the N-terminal domain (NTD) drives the physiologic self-assembly necessary for RNA binding through two canonical RNA recognition motifs, the C-terminal LCD, a region where most ALS/FTLD-associated mutations are found, mediates dysfunctional assemblies (24, 68–71). Segments of the TDP-43 LCD can form both steric zippers and LARKS. Interestingly, the NTD appears to resist self-assemblies regulated by pathologic (LCD) self-interactions (72). Similarly, tau protein can be divided into distinct motifs: the negatively charged NTD and CTD and the positively charged proline-rich domain (PRD) and microtubule-binding domain (MTBD) (Figure 1B). Six different tau isoforms are generated in the human brain by alternative splicing containing varying NTD inserts (zero, one, or two) and three or four MTBD repeats. Importantly, tau contains two aggregation-prone steric zipper motifs within the MTBD (10), and while both the PRD and MTBD domains are capable of phase separation, the PRD has a prominent role in regulating tau liquid-liquid phase separation in cells (42). Condensate assembly through heterotypic phase transitions Membrane-bound structures were historically considered the established systems of intracellular organization. However, emerging research has since highlighted the role of dynamic biomolecular condensates, commonly referred to as membraneless organelles, as another process underlying cellular compartmentalization. These are dynamic assemblies formed through phase transitions consisting of homotypic/heterotypic interactions between proteins, nucleic acids, and cofactors (26, 28, 36). Hundreds or thousands of intracellular biomolecular interactions (“heterotypic buffering effect”) that occur under physiologic conditions prevent deleterious homotypic protein interactions observed in NDDs (22, 28). Thus, a better understanding of liquid-like phases and their liquid-to-solid transitions is important for understanding NDD pathogenesis. A leading hypothesis for condensate assembly defines the condensate components as either scaffolds or clients. Scaffolds ultimately regulate the incorporation of various client biomolecules, which are not necessary for condensate assembly but essential for condensate dynamics and function. Subsequently, necessary scaffold-client interactions drive the assembly and tune the dynamic compositions of biomolecular condensates (33, 43, 73, 74). RNA species are integral components of many described condensates and, like proteins, are capable of scaffolding condensates through multivalent interactions (24, 75–83). Furthermore, RNA can promote or dissolve condensates scaffolded by RNA-binding proteins (RBPs), likely depending on their sequence, structure, and valence (23, 75, 83, 84). Scaffolding molecules encode structural elements that drive and regulate phase transitions, including the generation of pre-assemblies such as small clusters and/or liquid-like phases. Multivalence is a common feature of scaffolding molecules and acts as a critical regulator of heterotypic phase transitions (35, 39, 73, 74, 79). Multivalence can be achieved in several ways, though it generally involves weak, transient contacts through modular interaction domains. Scaffolding protein motifs participating in specific interactions can occur on folded domains, low-complexity motifs, and sometimes even single residues. These interaction motifs form reversible cross-links through various chemical interactions, referred to as stickers (73, 74, 85). Sticker motifs are the same cross-links that drive protein folding, fold-specific recognition motifs, and many “classic” molecular assemblies known in biology (74). Additionally, while stickers engage in physical cross-links, various spacer sequences within the protein impact its overall solubility. Biomolecular condensation can be coupled to both segregative phase separation (density transitions) and percolation, an associative phase transition (or networking transitions), as well as cooperative density-driven network transitions (phase separation coupled to percolation) (28). In a percolated network, physically cross-linked networks form via liquid-to-gel transitions, leading to network-spanning structures (28). The valence of stickers and their interaction strengths define the intrinsic concentration, or “percolation threshold” (Cperc), necessary for networking phase transitions (28, 74). Client-scaffold binding significantly alters saturation concentrations required for assembly and dissolution, providing switch-like, rapid behavior. Therefore, a networking transition is enabled by specific interactions between biomolecules (scaffolds) with a multivalence of interaction motifs (stickers). Regardless of the mechanisms driving their assembly, the ability to locally concentrate specific biomolecules is a classic description of all discovered biomolecular condensate structures (Figure 1C). Cellular functions of biomolecular condensates Today, biomolecular condensates are thought to spatially organize related processes in compartments ranging from the nucleus to the end of neuronal synapses (Figure 2A) (86–88). Primarily, condensates act as organization hubs, allowing spatiotemporal control of a variety of localized functions. They can also act as reaction crucibles, where the concentration of molecules in a condensed state promotes dynamic exchanges of products/reactants and sequesters biomolecules for storage or degradation. By spatiotemporally organizing biomolecules, unique biomolecular condensates dictate the biosynthesis, transport, regulation, and function of the basic building blocks necessary for cellular homeostasis (28, 30, 32, 36, 89, 90). In the absence of membrane-bound elements, distinct condensates can regulate nuclear functions, including chromatin compaction, DNA repair, RNA transcription, processing, transport, and decay (29, 89, 91–95). The most widely known subnuclear biomolecular condensate is the nucleolus. Nucleoli are multiphase condensates present within all eukaryotic organisms and are known as the site of rRNA transcription and ribosome assembly. The liquid-like state of nucleoli allows for a rapid exchange of newly transcribed/processed rRNA and ribosomal subunits between subcompartments of the nucleolus, permitting proper assembly and export of ribosomes from the nucleus (89, 94, 96–98). Phase-separated condensates are also implicated in driving gene activation through transcriptional condensates assembled at enhancer-rich gene clusters (95, 99–101). Properties inherent to chromatin, including the spacing of nucleosomes, allow it to phase-separate within the nucleoplasm, thus enabling the establishment and maintenance of distinct chromatin subcompartments (91, 102). Other well-studied nuclear condensates worth mentioning include Cajal bodies (associated with maturation of spliceosomal RNA and small nuclear ribonucleoprotein complexes), paraspeckles (involved in RNA editing and a protein buffering reservoir), nuclear speckles (“assembly line” involved in transcription-splicing mRNA export), and promyelocytic leukemia (PML) bodies (implicated in DNA damage and telomere maintenance) (89, 92). Another well-known biomolecular condensate is cytoplasmic stress granules (SGs). SGs are a considerable focus in the field of neurodegeneration following the discovery that several disease-linked RBPs, including TDP-43 and tau, can localize to and modify SG assembly and dynamics (103). This micrometer-sized condensate assembles RNA and RBPs under various cellular stressors, and these structures regulate RNA stability and triage non-essential protein translation until the stress is removed (103–109). Recent studies show that the initial pre-assembly of G3BP1/2 dimers (which promote liquid-liquid phase separation) and the newly released mRNAs from polysomes during translational inhibition provide a physical platform for SG assembly. This initial assembly process is then followed by the subsequent recruitment of client molecules required for SG condensation and function necessary during cellular stress (79, 109). During the last several years, studies demonstrate the importance of liquid-like condensation with regard to the spatiotemporal organization of neurons (86–88, 110). This growing group of structures includes synaptic active zones, synaptic vesicles, and excitatory/inhibitory pre- and postsynaptic densities (87, 111–114). Further, to maintain active signaling complexes necessary for electrical signaling homeostasis and physiologic function, neurons rely on localized protein translation in axons/dendrites/synapses, which can be up to 1 meter in distance from the cell body (28, 115). Notably, a fraction of the intracellular RNA is associated with RBPs in condensates termed ribonucleoprotein (RNP) granules. Once these silenced RNA granules arrive at axons/dendrites/synapses, signaling-dependent PTMs regulate condensate properties, resulting in the release of RNA for either degradation or translation; this is particularly important for maintaining dendritic plasticity and regulating axon growth, regeneration, and maintenance (45, 115–117). Tau and TDP-43 regulate biological processes within liquid-like condensates in various cellular compartments, from the nucleoplasm to the synapse (12, 76, 118–121). Interestingly, tau and TDP-43 share many functions, as revealed by an extensively similar interactome embodied by RNP complexes, RNA/protein metabolism, molecular transport, and the neuronal stress response (122–124). Tau is usually a cytosolic axonal protein, and under disease conditions, tau accumulates in postsynaptic compartments, presynaptic terminals, and the nucleus (10, 125–127). Physiologically, tau can undergo phase separation to enhance the polymerization of microtubules by condensing tubulin dimers (118, 120). This drives microtubule polymerization, after which tau dissipates onto the microtubule surface. TDP-43 exerts multiple functions, including the regulation of splicing, trafficking, and stabilization of RNA (40, 123, 128, 129). While TDP-43 typically resides in the nucleus, it also shuttles from the nucleus to the cytoplasm and is found mislocalized to the cytoplasm of diseased neurons (40, 128). Notably, TDP-43 is a component of several RNP granules, including paraspeckles, nuclear stress bodies, and RNA transport granules in neurons (12, 69, 130). Biomolecular condensate dysfunction Given the essential roles that biomolecular condensates have in regulating cellular processes, one expects that many condensates are dysregulated in related diseases. Current evidence suggests that condensate dysregulation is a prevalent pathogenic mechanism underlying a broad spectrum of human diseases best described across NDDs and cancer (26, 27, 131). NDD phenotypes resulting from aging/disease-related insults include genomic DNA damage, defects in nucleocytoplasmic transport, and altered protein and RNA homeostasis (3, 16, 132–135). Under such conditions, dysregulated gene expression, alternative splicing events, disrupted RNA/protein transport, abnormal RNA/protein PTMs, and a loss in RNA/protein quality control have been observed. Many of these changes will directly impact threshold concentrations for phase separation, resulting in aberrant compositions and potential loss- and gain-of-function toxicity mechanisms. Consistent with this, pathogenic mutations across NDDs and cancer are increasingly associated with condensate dysregulation (25, 27, 136–139). The relationship between pathogenic mutations and dysregulated condensates may be best understood by studying RBPs. Many RBPs, including TDP-43 and non-canonical RBPs like tau, are genetically linked to NDDs (22, 25, 83, 107, 140–143). Such mechanisms include enhanced driving forces for liquid-liquid phase separation and liquid-to-solid transitions, as well as altered material properties and localization of the condensates they reside within. Consistent with this notion, prolonged residency time within dense liquid-like phases was shown to increase the likelihood of liquid-to-solid phase transitions for tau, TDP-43, and other NDD-related proteins using in vitro model systems (20, 22, 23, 27, 68, 71, 108, 137, 139). However, disease-causing mutations in RBPs shift the balance of interactions between RNP assemblies, which, regardless of the mutation’s impact on liquid-to-solid phase transitions, ultimately alters condensate composition, material properties, and function (22, 23, 27, 29, 107, 144). This discovery has uncovered potentially novel mechanisms of toxicity and prompted a reexamination of loss- and gain-of-function mutations in solid-phase transitions (22, 27, 29, 38, 137, 145). Notably, the altered subcellular localization of critical condensate scaffolds can change the behavior of the scaffold and condensate components, leading to dysfunctional condensate assembly and toxicity. Additionally, disease-causing mutations may perturb the selective partitioning/exclusion of critical clients necessary for condensate assembly, localization, material properties, and subsequent function (Figure 2B). Collectively, these discoveries have led to an exciting new framework for understanding the cellular biology underlying, and the potential molecular mechanisms driving, NDDs. Future research into dysregulated soluble protein phases containing tau/TDP-43 and other pathologic proteins will likely reveal additional links between aberrant condensates and neurotoxic mechanisms. Targeting aberrant phase transitions Extensive knowledge regarding alterations to the localization and biophysical properties of tau and TDP-43 in disease has provided fundamental examples linking aberrant phase transition behaviors with toxicity and potential targets for therapeutic intervention. Review of current and potential therapeutic targeting strategies directed at tau and TDP-43 proteinopathies highlights three potential therapeutic avenues that utilize the phase transition– and condensate-based hypotheses of NDDs (Figure 3). We will first examine strategies that directly target tau and TDP-43 (Table 1). Based on the residency of these pathologic proteins within various biomolecular condensates or “pathologic condensates,” we will discuss unique strategies that leverage properties of condensate biology and the critical cellular pathways regulating biomolecular condensates (Table 2). Modify the pathologic protein Reduce cellular accumulation of NDD-associated proteins. As previously mentioned, protein phase transitions can be described by local saturation concentrations (Csat) and strongly influenced by protein concentration (28, 33). Therefore, it is unsurprising that both tau and TDP-43 overexpression in cellular and animal models results in neurodegeneration and is further exacerbated by disease-causing mutations (10, 146, 147). Thus, targeting RNA to reduce the cellular accumulation of NDD proteins, such as through GAPmer antisense oligonucleotides (ASOs), bypasses the many unresolved questions regarding the toxicity of specific protein conformations, modifications, and polymeric assemblies and effectively prevents downstream toxicity. For TDP-43, both motor deficits and embryonic lethality have been described after partial and complete knockdown in animal models, respectively (147, 148). Therefore, reduction of wild-type TDP-43 levels does not appear viable for clinical translation. Tau knockdown, however, has proven tolerable in many experimental models and repeatedly demonstrated cognitive protection in AD and FTD-taumut animal models (149–152). More recent work extends this protection to neuronal cultures treated with ALS synaptoneurosomes (153). Tau-lowering strategies include tau-targeting immunotherapies and RNA-targeting MAPT ASOs, which are currently in clinical trials for tauopathies (154). Additionally, MAPT isoform–specific ASOs and small molecules targeting MAPT RNA splicing regulatory elements have demonstrated therapeutic potential by targeting overabundant tau isoforms in rodent models of genetic forms of frontotemporal dementia (FTD) (155–157). DNA-targeting zinc finger protein transcription factors (ZFP-TFs) capable of directly targeting and lowering specific protein-coding sequences provide long-lasting reductions in tau expression following a viral-mediated introduction in disease models of tauopathy (151, 158). Embedded within most genes encoding IDPs, an endogenous mechanism exists that controls translation through the expression of natural antisense transcripts (NATs) that contain mammalian-wide interspersed repeats (MIRs) (159). These MIR-NAT sequences compete for rRNA pairing and transcript translation and may act as a potential avenue for therapeutic intervention. For example, silencing of the MIR-NAT MAPT-AS1 led to increased tau levels, and its expression correlated with aggregated tau in the human brain (159). Inhibit pathologic self-interactions. As discussed in previous sections, intrinsic and extrinsic factors govern the energy state of intramolecular interactions, orming physiologic protein conformations and preventing aggregation-prone conformations (5, 53, 62). Therefore, designing small molecules that stabilize physiologic protein conformations and prevent pathologic conformations, self-assembly, and subsequent deleterious phase transitions is a viable therapeutic strategy. Attempts to design IDR small-molecule modulators have proven difficult, and no clinically approved small-molecule therapeutics targeting disease-related IDPs/IDRs currently exist (160). However, studies did identify small molecules that recognize monomeric tau and TDP-43, thus supporting the possibility of this approach for future investigation (161–164). Tau monomers may occupy distinct conformational ensembles, where some conformations are relatively inert, while others have the intrinsic ability to self-assemble and are seed-competent (63, 165). The initiation of tau self-assembly likely begins with a stable transition of tau monomer from an inert to a seed-competent monomeric form. One of the most well-studied tau-interacting ligands, the small molecule methylene blue (MB) and its derivative TRx0237, has been through several phase III clinical trials (163, 166). MB and its derivatives directly interact with tau monomer, thus blocking tau-tau interactions to prevent and reverse tau aggregation in vitro (167, 168). Investigation of TRx0237 and other tau-binding small molecules highlights the potential of binding and sequestering IDPs in monomeric, soluble states. Similarly, a small molecule, nTRD22, targeting the N-terminal domain of TDP-43 was recently shown to be an allosteric modulator of TDP-43–RNA binding and conferred protection against motor deficits in an ALS-Drosophila model that overexpresses TDP-43 (169). These highlighted examples suggest that further research targeting monomeric forms of pathologic proteins with small molecules is a viable and promising approach to prevent and/or reverse aberrant phase transitions. Recent work by us and others also highlights the ability of specific RNAs to regulate protein phase transitions through specific RNA-protein interactions. In the case of TDP-43, homotypic low-complexity domain (LCD) interactions initiate its pathologic aggregation through aberrant liquid-liquid phase separation, and this homotypic interaction is antagonized by RNA binding (23, 24, 76). An RNA-dependent mechanism of pathologic interaction was also shown for other NDD-associated RBPs, including FUS and tau (120, 142, 170, 171). This mechanism highlights an intriguing RNA-based targeting strategy in which an RNA aptamer or “bait oligonucleotide” might be able to engage RNA-deficient TDP-43 in the cytoplasm and prevent or reverse pathologic phase transitions. In the case of TDP-43, a bait oligonucleotide (Clip_34) comprising the TARDBP mRNA 3′-UTR autoregulatory domain engages the TDP-43 RNA recognition motifs and prevents neurotoxic TDP-43 self-interactions, phase transitions, and associated in vitro neurotoxicity (24, 172). Immunotherapies to disrupt existing pathologic homotypic assemblies also showed promise for both tau and TDP-43 (161, 173–175). Tau-based immunotherapies have gone from proof-of-concept studies to clinical trials for AD and other tauopathies (154). Several notable disease-conformation-specific tau antibodies have since been developed, presenting promising results for reducing tau aggregation in preclinical models of tauopathy (176, 177). For example, the PNT001 antibody is capable of recognizing a toxic, trans-to-cis conformational change occurring early in tauopathies (178–180). PNT1001 prevents tau aggregation, neuropathology, and cognitive impairment in several preclinical tauopathy models, including models of CTE. PNT1001 is currently entering clinical trials in patients with various tauopathies, including traumatic brain injury (TBI). Similarly, a rationally developed antibody targeting an RNA recognition domain of TDP-43 was shown to successfully reduce insoluble TDP-43 inclusions, inflammation, and cognitive impairment in a transgenic ALS mouse model expressing the familial ALS TDP-43G348C protein (173). Soluble oligomeric protein assemblies of tau and TDP-43 are synaptotoxic and, in the case of tau, capable of propagating self-assembly through connected neural networks (46, 50, 54, 181–183). Recently, many rational designs leveraging stable structures mediated by LCDs/IDRs through aberrant phase transitions and the accumulation of homotypic self-assemblies have brought exciting opportunities for structure-specific targeting. Targeting the neurotoxic and misfolded protein structure and not the protein monomers should limit interference with the physiologic function of the protein when in its proper conformation. This is notable since the physiologic phase separation of IDR-containing proteins into biomolecular condensates is critical for various cellular processes. For example, physiologic phase transitions of TDP-43 into reversible biomolecular condensates is hypothesized to be essential for the binding of specific RNA sequences (23, 24, 76, 184). Thus, the development of strategies that target pathologic but not physiologic phase-separated assemblies is a powerful approach. Regarding tau, multiple in vitro studies identified small molecules that inhibit tau assembly with various mechanisms of action. These include molecules that block inducer-specific fibril growth, preventing fibril growth by initializing nontoxic, off-pathway assemblies, and those capable of disassembling preformed fibrils (185–188). Use of cryo–electron microscopic structures of human AD tau filaments bound to small molecules has allowed the identification of novel, drug-like molecules capable of disaggregating brain-derived tau fibrils in vitro (189). One example is the small molecule Anle138b, which is currently in clinical trials for Parkinson’s disease and multiple-system atrophy and has previously been shown to reduce tau aggregation and behavioral deficits in numerous cellular and animal models of tauopathy (188). Experimental evidence demonstrated that Anle138 avoids tau monomer binding and selectively binds oligomeric tau assemblies, preventing the formation of amyloidogenic fibrils (188). Furthermore, crystal structures of tau steric zippers led to the rational design of small steric zipper–binding peptides, referred to as “fibril capping” peptides (190). Modulate pathologic protein PTMs. PTMs, including covalent modifications and cleavage events, offer a fine-tuned response to diverse extracellular stimuli and intracellular signaling pathways (45, 191–193). PTMs substantially alter the intrinsic properties of a sequence and thus regulate intra- and intermolecular interactions (62). Therefore, covalent modifications can act as potent regulators of protein/RNA conformations and, consequently, the properties of biomolecular condensates. In disease, tau and TDP-43 are often found heavily modified by PTMs (phosphorylation, acetylation, ubiquitination, etc.) and cleaved into fragments (10, 53, 193–197). While the effect of PTMs on biomolecular phase behavior is only beginning to be understood, PTMs may directly regulate phase behavior by altering either intra- or intermolecular interactions, leading to an altered Csat. Lysine-modifying acetylation in tau and TDP-43 significantly reduces critical lysine-RNA interactions, resulting in altered phase behaviors (198–200). Targeting of tau acetylation after TBI using acetylation-inhibiting drugs (salsalate) is associated with reduced neurodegeneration in humans and prevents tau mislocalization, insolubility, and cognitive deficits in preclinical models (201). TDP-43 acetylation, which mitigates RNA binding, enhances its phase separation into complex nuclear droplets called anisomes that colocalize with HSP70 and can promote aberrant phase transitions when localized to the cytoplasm. This results in gel-like and insoluble assemblies and highlights the role of RNA binding as a modulator of TDP-43 liquid-liquid phase separation (24, 200, 202). Importantly, pairings of PTMs may have distinct effects on downstream modifications, either stimulating or inhibiting hallmark phase transitions (53, 197, 203, 204). PTM-modifying therapies will require extensive study with regard to the complex interplay between single and combinatorial PTMs and how PTMs alter biomolecular interacting partners, resulting phase behaviors, and subsequent neurotoxicity. Modify pathologic condensates Leverage heterotypic multivalent interactions. The growing knowledge regarding condensate assembly and regulation opens avenues for interfering with pathologic phase transitions. With the inherent limitations of direct targeting of pathogenic phase transitions of a single protein (tau or TDP-43), targeting biomolecular condensates that might drive aberrant phase transitions vastly extends the pool of drug targets. The residency and scaffolding potential of pathologic proteins in critical cellular condensates are intriguing. Modifying condensate scaffolds would significantly affect condensates’ stability, including assembly, dissolution, material properties, and composition of scaffold/ligands/etc. (28, 43, 73, 79, 205). Thus, disrupting specific components and regulatory pathways of biomolecular condensates to indirectly modify abnormal hallmark phase transitions and cellular toxicity may be a therapeutic approach. The ultimate goal of this is to shift tau or TDP-43 Csat and phase transition behaviors. Scaffold modulation can be achieved in several ways. Approaches may include preventing or stabilizing protein-protein, protein-RNA, and RNA-RNA interactions that contribute to condensate scaffolding. Intriguingly, the genetic manipulation of RBPs often alters the rate of tau and TDP-43 aggregation in several model systems (206–209). TIA1 is an RBP and a major component of stress granules (SGs). Previous studies indicate that TIA1 interacts with tau, and this interaction modulates tau aggregation and toxicity (208, 210, 211). TIA1 knockdown prevents tau-mediated toxicity, reduces toxic soluble tau oligomers, and increases insoluble tau fibrils (208). Importantly, tau fibrils isolated from the diseased brain contain numerous RNA species. Recent research has demonstrated tau-mediated disruptions in RNA metabolism, leading to tau-RNA accumulations building on the nuclear envelope (212, 213). Remarkably, promoting nonsense-mediated mRNA decay with a small molecule, tranilast, disrupts these tau-RNA accumulations, suppressing neurodegeneration and locomotor deficits in a tau-transgenic Drosophila model. Ataxin-2 (ATXN2) is an RBP found in mature SGs, and intermediate CAG expansions within the ATXN2 gene are found in subsets of ALS cases (214). Recent work found that ATXN2 knockdown reduces abnormal SG formation and is neuroprotective in both in vitro and in vivo rodent models with elevated levels of TDP-43 (209). Additionally, ATXN2 reduction significantly reduces TDP-43 pathology. Further, ALS’s most common genetic cause (expansions of C9orf72) leads to the overexpression of expanded GC RNA repeats, leading to TDP-43 mislocalization, assembly, and toxicity (215). Recently, a small-molecule-guided ribonuclease-targeting chimera (RIBOTAC) method capable of directly targeting the removal of G4C2 duplications prevented TDP-43 insolubility and neurotoxicity in animal models (216). Additionally, recent work has demonstrated that upregulating an endogenous TDP-43–interacting noncoding RNA, NEAT1_1, lowered TDP-43 insolubility and toxicity in Drosophila and yeast models of TDP-43 proteinopathy (217). Together, this suggests that pathogenic interactions within biomolecular condensates may promote aberrant TDP-43 and tau phase transitions and that modulating these interactions might confer neuroprotection and be a potential therapeutic approach. While considerable attention has been focused on protein modifications, recent work highlights a long list of covalent nucleic acid modifications that may alter TDP-43 and tau phase transitions (78, 218–222). DNA and RNA methylation are potent regulators of nucleic acid phase separation and affect the condensation properties of specific protein–nucleic acid complexes (221, 223, 224). Interestingly, the knockdown of the canonical RNA N6-methyladenosine (m6A) reader YTHDF2 was recently shown to prolong the survival of induced pluripotent stem cell human neurons carrying ALS-associated mutations (225). Consistent with this, knockdown of the canonical RNA m6A reader HNRNPA2B1 and the m6A writer METTL3 rescued tau-oligomer-induced neurodegeneration in models of tauopathy (206). Thus, the targeting of these RNA modifications is slowly being revealed as a novel approach capable of regulating pathologic protein phase transitions. Restore proteostatic networks. The proteostasis network, a protein quality control (PQC) system, regulates and balances protein synthesis, folding, transport, and degradation (226–228). Impairment of one or several PQC mechanisms can result in aberrant phase transitions and the accumulation of protein aggregates inside neurons. The PQC system is an integrated network of molecular chaperones, co-chaperones, and two degradative systems, the ubiquitin-proteasome system (UPS) and autophagy, a lysosome-mediated bulk degradation pathway (226, 229). Traditionally, autophagy was believed to preferentially clear protein aggregates with a certain amount of “liquidity” in a process referred to as aggrephagy (230–232). An arm of aggrephagy was recently discovered and selectively targets protein aggregates with little liquidity (solids) for lysosomal degradation, thus highlighting critical cellular mechanisms that interact with biomolecular condensates with specific intrinsic material properties (231). While aggrephagy was thought to process condensates with some liquidity, recent work demonstrated that the CCT2 autophagy receptor allows for the selective targeting of solid condensates. Notably, the upregulation of CCT2 cleared several solid protein aggregates from cells, including mutant tau protein (231). Several pharmacologic agents that modulate the ATPase activity of HSP70, a core chaperone, have been designed and tested in NDD models (233, 234). Interestingly, reduction of HSP70 ATPase activity transforms TDP-43 liquid phases into gel-like structures, leading to insoluble TDP-43 assemblies and increased toxicity (200). Substantial efforts found that non-core chaperones, including a specific class, the peptidyl-prolyl cis-trans isomerases (PPIases), protected against aberrant tau phase transitions (235). Specifically, Pin1 catalyzes proline cis-to-trans isomerization, a conformational change that protects against the stabilization of toxic conformations that lead to pathologic tau fibrils (176, 177, 179). Increasing evidence shows that nuclear-import receptors chaperone and disaggregate RBPs, including TDP-43 (40, 236, 237). Not only do nuclear localization sequences (NLSs) mediate the nuclear import of NLS-containing proteins, but they also inhibit deleterious phase transitions and promote the disaggregation of solid assemblies. In the cytoplasm, specific nuclear-import receptors that engage the TDP-43 NLS, importin-α and -β, prevent and reverse TDP-43 aggregation n models of C9orf72 ALS/FTLD (236). The UPS predominantly regulates soluble tau and TDP-43, and the accumulation of these species can lead to protein nucleation (238–242). While macroautophagy pathways can directly sequester and degrade larger condensates, soluble protein monomers can be degraded by chaperone-mediated autophagy (CMA). The inability to remove accumulating soluble proteins eventually promotes the aggregation of the CMA-regulated proteome. Consistent with this, CMA deficiency in the aging brain is an aggravating factor in the onset of NDD (243). Activation of CMA with small molecules has proven neuroprotective in animal models of tauopathy (243, 244). Modulate condensate physicochemistry. While the direct engagement of condensate components may allow a prospective drug to occupy a condensate, a drug may also concentrate within a condensate due to a network of transient contacts without high affinity toward a specific target (101, 245–249). Therefore, a small molecule, through interactions with the chemical environment of the condensate, may strongly influence condensate properties regulating the formation or dissolution of condensates. Therefore, using small-molecule ligands to target condensates may be a promising therapeutic strategy. This premise is clearly illustrated by cellular metabolites like ATP, cAMP, glucose, and many others, which were previously demonstrated to modulate condensate properties (34, 43, 73, 250–252). Several known small molecules can alter the phase behaviors of tau and TDP-43 proteins by either directly interfering with the ability of the pathologic protein to self-condense into liquid-like phase, or interfering with their recruitment to biomolecular condensates (i.e., SGs) (245–248). Specifically, molecules with planar moieties, such as mitoxantrone, were shown to prevent TDP-43 cytoplasmic localization and prolonged residency in SGs (246). Further, the compound myricetin can slow the liquid-like phase separation of tau, shifting its phase boundary while stabilizing the interaction of tau protein within the aggrephagy clearance pathway (249). Besides regulating the properties of existing hallmark condensates with small molecules, interest in generating artificial condensate systems to engage with endogenous condensates is growing. Interestingly, the cytoplasmic expression of the neuronal chaperone proSAAS created micron-scale membraneless spheres with condensate features that selectively encapsulated and sequestrated TDP-43 aggregates and reduced their toxicity in cell culture models (253). Further work is under way designing programmable condensates capable of sequestering pathologic aggregates, stabilizing the pathologic proteins’ normal physiology, and facilitating drug delivery and enrichment toward specific condensates. Conclusion It is believed that the biochemical changes responsible for initiating NDDs begin decades before the clinical presentation (2, 9, 26, 226, 228, 254). Furthermore, there are fundamental challenges to differentiating “normal” age-related events from pathologic biochemical processes that drive the earliest stages of neurodegeneration or distinguishing primary causes from a cascade of secondary insults. Aberrant protein conformations, oligomers, and fibrils composed of neuropathologic protein depositions may symbolize both a symptom and a cause of the underlying disease. As new discoveries emerge describing structure-specific protein assemblies in NDD subtypes, a thorough understanding of the cellular conditions driving these unique self-assemblies will prove important to develop disease-modifying therapies (56, 57, 64, 255). Condensate biology is fundamental to numerous cellular processes, and a growing understanding of these mechanisms is already transforming our understanding of how cells spatiotemporally organize biomolecules to regulate critical cell functions. As the formation of biomolecular condensates involves and influences all levels of macromolecular organization, condensate biology can profoundly expand our understanding of the pathologic conditions that lead to toxic protein assemblies, resulting downstream cellular dysfunction, and subsequent neurodegeneration. Targeting of aberrant phase transitions as a therapeutic intervention for neurodegenerative disorders will require substantial work to better characterize the diverse condensate subtypes and their components, physicochemical properties, assembly mechanisms, and physiologic function. Author contributions BTH and LX conceptualized and outlined the contents of this review, figures, and tables. BTH wrote the initial full draft with input from LX. BTH and LX both addressed reviewers’ comments. LX is a Tsinghua MD, PhD Scholar working in the CJD laboratory and supported by a partnership between the University of Pittsburgh School of Medicine and the Tsinghua University School of Medicine. The CJD laboratory is supported by grants from the NIH (R01NS127187, L30AG048607, R01NS105756), the Target ALS Foundation, the LiveLikeLou Center for ALS Research at the University of Pittsburgh Brain Institute, and LiveLikeLou at the Pittsburgh Foundation. Version 1 07/03/2023 Electronic publication Figure 1 Phase transitions of NDD-related proteins. (A) Domain structure and interaction features of TDP-43 and tau. TDP-43 contains three domains: an N-terminal domain (NTD) including a nuclear localization sequence (NLS); two RNA recognition motifs (RRM1 and RRM2); and a C-terminal domain (CTD) with a short α-helical fold (CR helix) and a glutamine/arginine-rich region (Q/N). Tau contains four domains: the negatively charged NTD and CTD, and the positively charged proline-rich domain (P1–P2) and microtubule-binding domain (MTBD; R1–R4). Six different tau isoforms are generated by alternative splicing containing zero, one, or two NTD inserts and three or four MTBD repeats. The intrinsically disordered regions (IDRs), aggregation-prone steric zippers, and domain-dependent homo/heterotypic biomolecular interactions of TDP-43 and tau are shown accordingly. LLPS, liquid-liquid phase separation. (B) Aberrant protein conformations, toxic polymeric self-assemblies, and solid accumulations of proteins are found across the most common NDDs. TDP-43 and tau are modular, multivalent proteins exhibiting conformational flexibility, allowing diverse monomeric conformations, polymeric assemblies, and liquid-like phase behaviors in normal physiology and pathology. Sequence-specific properties found within distinct protein domains (modular interaction domains, intrinsically disordered regions, and amyloid-forming regions) are influenced by intrinsic (isoforms, mutations, PTMs) and extrinsic factors (molecular interactions, environmental conditions), ultimately regulating phase behavior and unique polymerization pathways. While increased homotypic interactions drive protein self-polymerization and the phase separation of proteins into liquid-like droplets, they are independent processes regulated by overlapping conditions. (C) TDP-43 and tau reside within multicomponent biomolecular condensates and thus are subjected to diverse homo/heterotypic biomolecular interactions, ultimately regulating physiologic and pathologic phase transitions. Biomolecules necessary for condensate assembly (scaffolds) spatially organize and concentrate functionally related biomolecules (clients) through liquid-like phase transitions. A sticker and spacer model has been proposed in which sticker sequences regulate multivalent networking interactions and spacer sequences regulate the solubilities of individual biomolecules and emerging networks. Figure 2 Hallmarks of neurodegeneration involve functions related to diverse biomolecular condensates. (A) Schematic diagram showing the localization of various biomolecular condensates in a neuronal cell. Various biomolecular condensates are associated with many cellular processes that influence the homeostasis of nucleic acids and proteins from the nucleus to the end of synapses. (B) Neurodegeneration is accompanied by genetic, transcriptomic, and translational disruptions within vulnerable, cell type–specific neuronal populations. Imbalances in nucleic acid and protein homeostasis will directly affect the compositions, localization, and function of condensates (loss of function), additionally leading to aberrant phase transitions occurring within pathologic condensates (gain of function). Additionally, pathologic protein assemblies lead to downstream disruptions of physiologic condensates. RI, type I regulatory subunity of cAMP-dependent protein kinase (PKA). Figure 3 Drug discovery avenues for targeting aberrant phase transitions associated with neurodegeneration. Three major avenues for targeting pathologic protein phase transitions in NDDs are proposed. (A) Modify the pathologic protein. The phase behavior of a pathologic protein may be directly modified by modulation of pathologic protein levels and PTMs, and by direct targeting of toxic homotypic interactions. (B) Modify the pathologic condensate. With the inherent limitations of direct targeting of a single protein, modifying pathologic condensates vastly extends the pool of drug targets. The aberrant condensate features may be altered by leveraging of heterotypic multivalent interactions and physicochemical properties, and by restoration of cellular proteostatic networks. Table 2 Multicomponent condensates containing tau and TDP-43 can be modified to prevent neurodegeneration Table 1 Tau and TDP-43 proteins can be directly targeted to prevent aberrant phase transitions and neurodegeneration Conflict of interest: CJD is cofounder of Confluence Therapeutics Inc. Copyright: © 2023, Hurtle et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License. Reference information: J Clin Invest. 2023;133(13):e168549. https://doi.org/10.1172/JCI168549. ==== Refs 1 Fu H et al Selective vulnerability in neurodegenerative diseases Nat Neurosci 2018 21 10 1350 1358 10.1038/s41593-018-0221-2 30250262 2 Elahi FM Miller BL A clinicopathological approach to the diagnosis of dementia Nat Rev Neurol 2017 13 8 457 476 10.1038/nrneurol.2017.96 28708131 3 Wilson DM et al Hallmarks of neurodegenerative diseases Cell 2023 186 4 693 714 10.1016/j.cell.2022.12.032 36803602 4 Eisenberg D Jucker M The amyloid state of proteins in human diseases Cell 2012 148 6 1188 1203 10.1016/j.cell.2012.02.022 22424229 5 Soto C Pritzkow S Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases Nat Neurosci 2018 21 10 1332 1340 10.1038/s41593-018-0235-9 30250260 6 Bellenguez C et al New insights into the genetic etiology of Alzheimer’s disease and related dementias Nat Genet 2022 54 4 412 436 10.1038/s41588-022-01024-z 35379992 7 Gan L et al Converging pathways in neurodegeneration, from genetics to mechanisms Nat Neurosci 2018 21 10 1300 1309 10.1038/s41593-018-0237-7 30258237 8 Karch CM et al Selective genetic overlap between amyotrophic lateral sclerosis and diseases of the frontotemporal dementia spectrum JAMA Neurol 2018 75 7 860 875 10.1001/jamaneurol.2018.0372 29630712 9 Arvanitakis Z et al Diagnosis and management of dementia: review JAMA 2019 322 16 1589 1599 10.1001/jama.2019.4782 31638686 10 Wang Y Mandelkow E Tau in physiology and pathology Nat Rev Neurosci 2016 17 1 5 21 10.1038/nrn.2015.1 26631930 11 Chang CW et al Tau: enabler of diverse brain disorders and target of rapidly evolving therapeutic strategies Science 2021 371 6532 eabb8255 10.1126/science.abb8255 33632820 12 Portz B et al FUS and TDP-43 phases in health and disease Trends Biochem Sci 2021 46 7 550 563 10.1016/j.tibs.2020.12.005 33446423 13 Zbinden A et al Phase separation and neurodegenerative diseases: a disturbance in the force Dev Cell 2020 55 1 45 68 10.1016/j.devcel.2020.09.014 33049211 14 Nelson PT et al Limbic-predominant age-related TDP-43 encephalopathy (LATE): consensus working group report Brain 2019 142 6 1503 1527 10.1093/brain/awz099 31039256 15 Chornenkyy Y et al Tau and TDP-43 proteinopathies: kindred pathologic cascades and genetic pleiotropy Lab Invest 2019 99 7 993 1007 10.1038/s41374-019-0196-y 30742063 16 Ling SC et al Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis Neuron 2013 79 3 416 438 10.1016/j.neuron.2013.07.033 23931993 17 Chung DEC et al Cellular and pathological heterogeneity of primary tauopathies Mol Neurodegener 2021 16 1 57 10.1186/s13024-021-00476-x 34425874 18 McKee AC et al TDP-43 proteinopathy and motor neuron disease in chronic traumatic encephalopathy J Neuropathol Exp Neurol 2010 69 9 918 929 10.1097/NEN.0b013e3181ee7d85 20720505 19 Tremblay C et al Accumulation of transactive response DNA binding protein 43 in mild cognitive impairment and Alzheimer disease J Neuropathol Exp Neurol 2011 70 9 788 798 10.1097/NEN.0b013e31822c62cf 21865887 20 Wegmann S et al Tau protein liquid-liquid phase separation can initiate tau aggregation EMBO J 2018 37 7 e98049 10.15252/embj.201798049 29472250 21 Ambadipudi S et al Liquid-liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau Nat Commun 2017 8 1 275 10.1038/s41467-017-00480-0 28819146 22 Mathieu C et al Beyond aggregation: pathological phase transitions in neurodegenerative disease Science 2020 370 6512 56 60 10.1126/science.abb8032 33004511 23 Hallegger M et al TDP-43 condensation properties specify its RNA-binding and regulatory repertoire Cell 2021 184 18 4680 4696 10.1016/j.cell.2021.07.018 34380047 24 Mann JR et al RNA binding antagonizes neurotoxic phase transitions of TDP-43 Neuron 2019 102 2 321 338 10.1016/j.neuron.2019.01.048 30826182 25 Nedelsky NB Taylor JP Bridging biophysics and neurology: aberrant phase transitions in neurodegenerative disease Nat Rev Neurol 2019 15 5 272 286 10.1038/s41582-019-0157-5 30890779 26 Alberti S Hyman AA Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing Nat Rev Mol Cell Biol 2021 22 3 196 213 10.1038/s41580-020-00326-6 33510441 27 Alberti S Dormann D Liquid-liquid phase separation in disease Annu Rev Genet 2019 53 171 194 10.1146/annurev-genet-112618-043527 31430179 28 Mittag T Pappu RV A conceptual framework for understanding phase separation and addressing open questions and challenges Mol Cell 2022 82 12 2201 2214 10.1016/j.molcel.2022.05.018 35675815 29 Shin Y Brangwynne CP Liquid phase condensation in cell physiology and disease Science 2017 357 6357 eaaf4382 10.1126/science.aaf4382 28935776 30 Li P et al Phase transitions in the assembly of multivalent signalling proteins Nature 2012 483 7389 336 340 10.1038/nature10879 22398450 31 Brangwynne CP et al Polymer physics of intracellular phase transitions Nat Phys 2015 11 11 899 904 10.1038/nphys3532 32 Lyon AS et al A framework for understanding the functions of biomolecular condensates across scales Nat Rev Mol Cell Biol 2021 22 3 215 235 10.1038/s41580-020-00303-z 33169001 33 Shimobayashi SF et al Nucleation landscape of biomolecular condensates Nature 2021 599 7885 503 506 10.1038/s41586-021-03905-5 34552246 34 Snead WT Gladfelter AS The control centers of biomolecular phase separation: how membrane surfaces, PTMs, and active processes regulate condensation Mol Cell 2019 76 2 295 305 10.1016/j.molcel.2019.09.016 31604601 35 Banani SF et al Compositional control of phase-separated cellular bodies Cell 2016 166 3 651 663 10.1016/j.cell.2016.06.010 27374333 36 Banani SF et al Biomolecular condensates: organizers of cellular biochemistry Nat Rev Mol Cell Biol 2017 18 5 285 298 10.1038/nrm.2017.7 28225081 37 Uversky VN et al Intrinsically disordered proteins in human diseases: introducing the D2 concept Annu Rev Biophys 2008 37 215 246 10.1146/annurev.biophys.37.032807.125924 18573080 38 Darling AL et al Intrinsic disorder-based emergence in cellular biology: physiological and pathological liquid-liquid phase transitions in cells Polymers (Basel) 2019 11 6 990 10.3390/polym11060990 31167414 39 Peran I Mittag T Molecular structure in biomolecular condensates Curr Opin Struct Biol 2020 60 17 26 10.1016/j.sbi.2019.09.007 31790873 40 Tziortzouda P et al Triad of TDP43 control in neurodegeneration: autoregulation, localization and aggregation Nat Rev Neurosci 2021 22 4 197 208 10.1038/s41583-021-00431-1 33654312 41 Wang X et al The proline-rich domain and the microtubule binding domain of protein tau acting as RNA binding domains Protein Pept Lett 2006 13 7 679 685 10.2174/092986606777790566 17018010 42 Zhang X et al The proline-rich domain promotes Tau liquid-liquid phase separation in cells J Cell Biol 2020 219 11 e202006054 10.1083/jcb.202006054 32997736 43 Ruff KM et al Polyphasic linkage and the impact of ligand binding on the regulation of biomolecular condensates Biophys Rev (Melville) 2021 2 2 021302 10.1063/5.0050059 34179888 44 Boija A et al Biomolecular condensates and cancer Cancer Cell 2021 39 2 174 192 10.1016/j.ccell.2020.12.003 33417833 45 Hofweber M Dormann D Friend or foe—post-translational modifications as regulators of phase separation and RNP granule dynamics J Biol Chem 2019 294 18 7137 7150 10.1074/jbc.TM118.001189 30587571 46 Peng C et al Protein transmission in neurodegenerative disease Nat Rev Neurol 2020 16 4 199 212 10.1038/s41582-020-0333-7 32203399 47 Chiti F Dobson CM Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade Annu Rev Biochem 2017 86 27 68 10.1146/annurev-biochem-061516-045115 28498720 48 Knowles TPJ et al The amyloid state and its association with protein misfolding diseases Nat Rev Mol Cell Biol 2014 15 6 384 396 10.1038/nrm3810 24854788 49 Dujardin S et al Tau molecular diversity contributes to clinical heterogeneity in Alzheimer’s disease Nat Med 2020 26 8 1256 1263 10.1038/s41591-020-0938-9 32572268 50 Gibbons GS et al Mechanisms of cell-to-cell transmission of pathological tau: a review JAMA Neurol 2019 76 1 101 108 10.1001/jamaneurol.2018.2505 30193298 51 Fitzpatrick AWP et al Cryo-EM structures of tau filaments from Alzheimer’s disease Nature 2017 547 7662 185 190 10.1038/nature23002 28678775 52 Cao Q et al Cryo-EM structures of four polymorphic TDP-43 amyloid cores Nat Struct Mol Biol 2019 26 7 619 627 10.1038/s41594-019-0248-4 31235914 53 Arakhamia T et al Posttranslational modifications mediate the structural diversity of tauopathy strains Cell 2020 180 4 633 644 10.1016/j.cell.2020.01.027 32032505 54 Fang YS et al Full-length TDP-43 forms toxic amyloid oligomers that are present in frontotemporal lobar dementia-TDP patients Nat Commun 2014 5 4824 10.1038/ncomms5824 25215604 55 Lye YS Chen Y-R TAR DNA-binding protein 43 oligomers in physiology and pathology IUBMB Life 2022 74 8 794 811 10.1002/iub.2603 35229461 56 Li D Liu C Hierarchical chemical determination of amyloid polymorphs in neurodegenerative disease Nat Chem Biol 2021 17 3 237 245 10.1038/s41589-020-00708-z 33432239 57 Shi Y et al Structure-based classification of tauopathies Nature 2021 598 7880 359 363 10.1038/s41586-021-03911-7 34588692 58 Arseni D et al Structure of pathological TDP-43 filaments from ALS with FTLD Nature 2022 601 7891 139 143 10.1038/s41586-021-04199-3 34880495 59 Laferrière F et al TDP-43 extracted from frontotemporal lobar degeneration subject brains displays distinct aggregate assemblies and neurotoxic effects reflecting disease progression rates Nat Neurosci 2019 22 1 65 77 10.1038/s41593-018-0294-y 30559480 60 Hughes MP et al Prevalence and species distribution of the low-complexity, amyloid-like, reversible, kinked segment structural motif in amyloid-like fibrils J Biol Chem 2021 297 4 101194 10.1016/j.jbc.2021.101194 34537246 61 Guenther EL et al Atomic structures of TDP-43 LCD segments and insights into reversible or pathogenic aggregation Nat Struct Mol Biol 2018 25 6 463 471 10.1038/s41594-018-0064-2 29786080 62 Houben B et al Protein structure and aggregation: a marriage of necessity ruled by aggregation gatekeepers Trends Biochem Sci 2022 47 3 194 205 10.1016/j.tibs.2021.08.010 34561149 63 Eschmann NA et al Signature of an aggregation-prone conformation of tau Sci Rep 2017 7 44739 10.1038/srep44739 28303942 64 Fitzpatrick AW Saibil HR Cryo-EM of amyloid fibrils and cellular aggregates Curr Opin Struct Biol 2019 58 34 42 10.1016/j.sbi.2019.05.003 31200186 65 Staderini T et al Biophysical characterization of full-length TAR DNA-binding protein (TDP-43) phase separation Protein Sci 2022 e4509 12 e4509 10.1002/pro.4509 36371546 66 Li HR et al The physical forces mediating self-association and phase-separation in the C-terminal domain of TDP-43 Biochim Biophys Acta Proteins Proteom 2018 1866 2 214 223 10.1016/j.bbapap.2017.10.001 28988034 67 Babinchak WM et al The role of liquid-liquid phase separation in aggregation of the TDP-43 low-complexity domain J Biol Chem 2019 294 16 6306 6317 10.1074/jbc.RA118.007222 30814253 68 Conicella AE et al ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain Structure 2016 24 9 1537 1549 10.1016/j.str.2016.07.007 27545621 69 Alami NH et al Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations Neuron 2014 81 3 536 543 10.1016/j.neuron.2013.12.018 24507191 70 Prasad A et al Molecular mechanisms of TDP-43 misfolding and pathology in amyotrophic lateral sclerosis Front Mol Neurosci 2019 12 25 10.3389/fnmol.2019.00025 30837838 71 Gopal PP et al Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons Proc Natl Acad Sci U S A 2017 114 12 E2466 E2475 10.1073/pnas.1614462114 28265061 72 Zeng YT et al Different intermolecular interactions drive nonpathogenic liquid-liquid phase separation and potentially pathogenic fibril formation by TDP-43 Int J Mol Sci 2022 23 23 15227 10.3390/ijms232315227 36499553 73 Ruff KM et al Ligand effects on phase separation of multivalent macromolecules Proc Natl Acad Sci U S A 2021 118 10 e2017184118 10.1073/pnas.2017184118 33653957 74 Choi J-M et al Physical principles underlying the complex biology of intracellular phase transitions Annu Rev Biophys 2020 49 107 133 10.1146/annurev-biophys-121219-081629 32004090 75 Van Treeck B Parker R Emerging roles for intermolecular RNA-RNA interactions in RNP assemblies Cell 2018 174 4 791 802 10.1016/j.cell.2018.07.023 30096311 76 Grese ZR et al Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties EMBO Rep 2021 22 12 e53632 10.15252/embr.202153632 34787357 77 Garcia-Jove Navarro M et al RNA is a critical element for the sizing and the composition of phase-separated RNA-protein condensates Nat Commun 2019 10 1 3230 10.1038/s41467-019-11241-6 31324804 78 Lewis CJT et al RNA modifications and structures cooperate to guide RNA-protein interactions Nat Rev Mol Cell Biol 2017 18 3 202 210 10.1038/nrm.2016.163 28144031 79 Sanders DW et al Competing protein-RNA interaction networks control multiphase intracellular organization Cell 2020 181 2 306 324 10.1016/j.cell.2020.03.050 32302570 80 Van Treeck B et al RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome Proc Natl Acad Sci U S A 2018 115 11 2734 2739 10.1073/pnas.1800038115 29483269 81 Roden C Gladfelter AS RNA contributions to the form and function of biomolecular condensates Nat Rev Mol Cell Biol 2021 22 3 183 195 10.1038/s41580-020-0264-6 32632317 82 Aarum J et al Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates EMBO Rep 2020 21 10 e49585 10.15252/embr.201949585 32945072 83 Mann JR Donnelly CJ RNA modulates physiological and neuropathological protein phase transitions Neuron 2021 109 17 2663 2681 10.1016/j.neuron.2021.06.023 34297914 84 Zhang X et al RNA stores tau reversibly in complex coacervates PLoS Biol 2017 15 7 e2002183 10.1371/journal.pbio.2002183 28683104 85 Martin EW et al Valence and patterning of aromatic residues determine the phase behavior of prion-like domains Science 2020 367 6478 694 699 10.1126/science.aaw8653 32029630 86 Wu X et al Liquid-liquid phase separation in neuronal development and synaptic signaling Dev Cell 2020 55 1 18 29 10.1016/j.devcel.2020.06.012 32726576 87 McDonald NA et al Assembly of synaptic active zones requires phase separation of scaffold molecules Nature 2020 588 7838 454 458 10.1038/s41586-020-2942-0 33208945 88 Ryan VH Fawzi NL Physiological, pathological, and targetable membraneless organelles in neurons Trends Neurosci 2019 42 10 693 708 10.1016/j.tins.2019.08.005 31493925 89 Lacroix E Audas TE Keeping up with the condensates: the retention, gain, and loss of nuclear membrane-less organelles Front Mol Biosci 2022 9 998363 10.3389/fmolb.2022.998363 36203874 90 Su Q et al Liquid-liquid phase separation: orchestrating cell signaling through time and space Mol Cell 2021 81 20 4137 4146 10.1016/j.molcel.2021.09.010 34619090 91 Shin Y et al Liquid nuclear condensates mechanically sense and restructure the genome Cell 2018 175 6 1481 1491 10.1016/j.cell.2018.10.057 30500535 92 Sabari BR et al Biomolecular condensates in the nucleus Trends Biochem Sci 2020 45 11 961 977 10.1016/j.tibs.2020.06.007 32684431 93 Ishov AM et al Coordination of transcription, processing, and export of highly expressed RNAs by distinct biomolecular condensates Emerg Top Life Sci 2020 4 3 281 291 10.1042/ETLS20190160 32338276 94 Frottin F et al The nucleolus functions as a phase-separated protein quality control compartment Science 2019 365 6451 342 347 10.1126/science.aaw9157 31296649 95 Henninger JE et al RNA-mediated feedback control of transcriptional condensates Cell 2021 184 1 207 225 10.1016/j.cell.2020.11.030 33333019 96 Lafontaine DLJ et al The nucleolus as a multiphase liquid condensate Nat Rev Mol Cell Biol 2021 22 3 165 182 10.1038/s41580-020-0272-6 32873929 97 Audas TE et al Immobilization of proteins in the nucleolus by ribosomal intergenic spacer noncoding RNA Mol Cell 2012 45 2 147 157 10.1016/j.molcel.2011.12.012 22284675 98 Feric M et al Coexisting liquid phases underlie nucleolar subcompartments Cell 2016 165 7 1686 1697 10.1016/j.cell.2016.04.047 27212236 99 Sharp PA et al RNA in formation and regulation of transcriptional condensates RNA 2022 28 1 52 57 10.1261/rna.078997.121 34772787 100 Guo YE et al Pol II phosphorylation regulates a switch between transcriptional and splicing condensates Nature 2019 572 7770 543 548 10.1038/s41586-019-1464-0 31391587 101 Klein IA et al Partitioning of cancer therapeutics in nuclear condensates Science 2020 368 6497 1386 1392 10.1126/science.aaz4427 32554597 102 Gibson BA et al Organization of chromatin by intrinsic and regulated phase separation Cell 2019 179 2 470 484 10.1016/j.cell.2019.08.037 31543265 103 Wolozin B Ivanov P Stress granules and neurodegeneration Nat Rev Neurosci 2019 20 11 649 666 10.1038/s41583-019-0222-5 31582840 104 Molliex A et al Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization Cell 2015 163 1 123 133 10.1016/j.cell.2015.09.015 26406374 105 Glauninger H et al Stressful steps: progress and challenges in understanding stress-induced mRNA condensation and accumulation in stress granules Mol Cell 2022 82 14 2544 2556 10.1016/j.molcel.2022.05.014 35662398 106 Gasset-Rosa F et al Cytoplasmic TDP-43 de-mixing independent of stress granules drives inhibition of nuclear import, loss of nuclear TDP-43, and cell death Neuron 2019 102 2 339 357 10.1016/j.neuron.2019.02.038 30853299 107 Mackenzie IR et al TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics Neuron 2017 95 4 808 816 10.1016/j.neuron.2017.07.025 28817800 108 Markmiller S et al Context-dependent and disease-specific diversity in protein interactions within stress granules Cell 2018 172 3 590 604 10.1016/j.cell.2017.12.032 29373831 109 Yang P et al G3BP1 is a tunable switch that triggers phase separation to assemble stress granules Cell 2020 181 2 325 345 10.1016/j.cell.2020.03.046 32302571 110 Hayashi Y et al Liquid-liquid phase separation in physiology and pathophysiology of the nervous system J Neurosci 2021 41 5 834 844 10.1523/JNEUROSCI.1656-20.2020 33472825 111 Milovanovic D De Camilli P Synaptic vesicle clusters at synapses: a distinct liquid phase? Neuron 2017 93 5 995 1002 10.1016/j.neuron.2017.02.013 28279363 112 Wu X et al Interactions between membraneless condensates and membranous organelles at the presynapse: a phase separation view of synaptic vesicle cycle J Mol Biol 2022 167629 1 167629 10.1016/j.jmb.2022.167629 35595170 113 Milovanovic D et al A liquid phase of synapsin and lipid vesicles Science 2018 361 6402 604 607 10.1126/science.aat5671 29976799 114 Zeng M et al Phase transition in postsynaptic densities underlies formation of synaptic complexes and synaptic plasticity Cell 2016 166 5 1163 1175 10.1016/j.cell.2016.07.008 27565345 115 Bourke AM et al De-centralizing the central dogma: mRNA translation in space and time Mol Cell 2023 83 3 452 468 10.1016/j.molcel.2022.12.030 36669490 116 Parker DM et al It’s just a phase: exploring the relationship between mRNA, biomolecular condensates, and translational control Front Genet 2022 13 931220 10.3389/fgene.2022.931220 35832192 117 Kim TH et al Phospho-dependent phase separation of FMRP and CAPRIN1 recapitulates regulation of translation and deadenylation Science 2019 365 6455 825 829 10.1126/science.aax4240 31439799 118 Tan R et al Microtubules gate tau condensation to spatially regulate microtubule functions Nat Cell Biol 2019 21 9 1078 1085 10.1038/s41556-019-0375-5 31481790 119 Hernández-Vega A et al Local nucleation of microtubule bundles through tubulin concentration into a condensed tau phase Cell Rep 2017 20 10 2304 2312 10.1016/j.celrep.2017.08.042 28877466 120 Hochmair J et al Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of tau condensates EMBO J 2022 41 11 e108882 10.15252/embj.2021108882 35298090 121 Maina MB et al The involvement of tau in nucleolar transcription and the stress response Acta Neuropathol Commun 2018 6 1 70 10.1186/s40478-018-0565-6 30064522 122 Tracy TE et al Tau interactome maps synaptic and mitochondrial processes associated with neurodegeneration Cell 2022 185 4 712 728 10.1016/j.cell.2021.12.041 35063084 123 Freibaum BD et al Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery J Proteome Res 2010 9 2 1104 1120 10.1021/pr901076y 20020773 124 Kavanagh T et al Tau interactome and RNA binding proteins in neurodegenerative diseases Mol Neurodegener 2022 17 1 66 10.1186/s13024-022-00572-6 36253823 125 Ittner A Ittner LM Dendritic tau in Alzheimer’s disease Neuron 2018 99 1 13 27 10.1016/j.neuron.2018.06.003 30001506 126 DeVos SL et al Synaptic tau seeding precedes tau pathology in human Alzheimer’s disease brain Front Neurosci 2018 12 267 10.3389/fnins.2018.00267 29740275 127 McInnes J et al Synaptogyrin-3 mediates presynaptic dysfunction induced by tau Neuron 2018 97 4 823 835 10.1016/j.neuron.2018.01.022 29398363 128 Lee EB et al Gains or losses: molecular mechanisms of TDP43-mediated neurodegeneration Nat Rev Neurosci 2012 13 1 38 50 10.1038/nrn3121 22127299 129 Afroz T et al Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation Nat Commun 2017 8 1 45 10.1038/s41467-017-00062-0 28663553 130 Sekar D et al TDP-43 and NEAT long non-coding RNA: roles in neurodegenerative disease Front Cell Neurosci 2022 16 954912 10.3389/fncel.2022.954912 36385948 131 Banani SF et al Genetic variation associated with condensate dysregulation in disease Dev Cell 2022 57 14 1776 1788 10.1016/j.devcel.2022.06.010 35809564 132 Hou Y et al Ageing as a risk factor for neurodegenerative disease Nat Rev Neurol 2019 15 10 565 581 10.1038/s41582-019-0244-7 31501588 133 Soto-Palma C et al Epigenetics, DNA damage, and aging J Clin Invest 2022 132 16 158446 10.1172/JCI158446 35968782 134 Donnelly CJ et al Aberrant RNA homeostasis in amyotrophic lateral sclerosis: potential for new therapeutic targets? Neurodegener Dis Manag 2014 4 6 417 437 10.2217/nmt.14.36 25531686 135 Spead O et al Nuclear pore dysfunction in neurodegeneration Neurotherapeutics 2022 19 4 1050 1060 10.1007/s13311-022-01293-w 36070178 136 Mehta S Zhang J Liquid-liquid phase separation drives cellular function and dysfunction in cancer Nat Rev Cancer 2022 22 4 239 252 10.1038/s41568-022-00444-7 35149762 137 Tsang B et al Phase separation as a missing mechanism for interpretation of disease mutations Cell 2020 183 7 1742 1756 10.1016/j.cell.2020.11.050 33357399 138 Zhang H et al Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases Sci China Life Sci 2020 63 7 953 985 10.1007/s11427-020-1702-x 32548680 139 Zhou X et al Mutations linked to neurological disease enhance self-association of low-complexity protein sequences Science 2022 377 6601 eabn5582 10.1126/science.abn5582 35771920 140 Nedelsky NB Taylor JP Pathological phase transitions in ALS-FTD impair dynamic RNA-protein granules RNA 2022 28 1 97 113 10.1261/rna.079001.121 34706979 141 Kim HJ et al Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS Nature 2013 495 7442 467 473 10.1038/nature11922 23455423 142 Maharana S et al RNA buffers the phase separation behavior of prion-like RNA binding proteins Science 2018 360 6391 918 921 10.1126/science.aar7366 29650702 143 Maziuk B et al Dysregulation of RNA binding protein aggregation in neurodegenerative disorders Front Mol Neurosci 2017 10 89 10.3389/fnmol.2017.00089 28420962 144 Niaki AG et al Loss of dynamic RNA interaction and aberrant phase separation induced by two distinct types of ALS/FTD-linked FUS mutations Mol Cell 2020 77 1 82 94 10.1016/j.molcel.2019.09.022 31630970 145 Aguzzi A Altmeyer M Phase separation: linking cellular compartmentalization to disease Trends Cell Biol 2016 26 7 547 558 10.1016/j.tcb.2016.03.004 27051975 146 Joel Z et al Improving mouse models for dementia. Are all the effects in tau mouse models due to overexpression? Cold Spring Harb Symp Quant Biol 2018 83 151 161 10.1101/sqb.2018.83.037531 30745408 147 Wood A et al Molecular mechanisms underlying TDP-43 pathology in cellular and animal models of ALS and FTLD Int J Mol Sci 2021 22 9 4705 10.3390/ijms22094705 33946763 148 Brown AL et al TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A Nature 2022 603 7899 131 137 10.1038/s41586-022-04436-3 35197628 149 DeVos SL et al Tau reduction in the presence of amyloid-β prevents tau pathology and neuronal death in vivo Brain 2018 141 7 2194 2212 10.1093/brain/awy117 29733334 150 Tai C et al Tau reduction prevents key features of autism in mouse models Neuron 2020 106 3 421 437 10.1016/j.neuron.2020.01.038 32126198 151 Roberson ED et al Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer’s disease mouse model Science 2007 316 5825 750 754 10.1126/science.1141736 17478722 152 DeVos SL et al Tau reduction prevents neuronal loss and reverses pathological tau deposition and seeding in mice with tauopathy Sci Transl Med 2017 9 374 eaag0481 10.1126/scitranslmed.aag0481 28123067 153 Petrozziello T et al Targeting tau mitigates mitochondrial fragmentation and oxidative stress in amyotrophic lateral sclerosis Mol Neurobiol 2022 59 1 683 702 10.1007/s12035-021-02557-w 34757590 154 Jabbari E Duff KE Tau-targeting antibody therapies: too late, wrong epitope or wrong target? Nat Med 2021 27 8 1341 1342 10.1038/s41591-021-01465-9 34385709 155 Chen JL et al Design, optimization, and study of small molecules that target tau pre-mRNA and affect splicing J Am Chem Soc 2020 142 19 8706 8727 10.1021/jacs.0c00768 32364710 156 Schoch KM et al Increased 4R-tau induces pathological changes in a human-tau mouse model Neuron 2016 90 5 941 947 10.1016/j.neuron.2016.04.042 27210553 157 Espíndola SL et al Modulation of tau isoforms imbalance precludes tau pathology and cognitive decline in a mouse model of tauopathy Cell Rep 2018 23 3 709 715 10.1016/j.celrep.2018.03.079 29669277 158 Wegmann S et al Persistent repression of tau in the brain using engineered zinc finger protein transcription factors Sci Adv 2021 7 12 eabe1611 10.1126/sciadv.abe1611 33741591 159 Simone R et al MIR-NATs repress MAPT translation and aid proteostasis in neurodegeneration Nature 2021 594 7861 117 123 10.1038/s41586-021-03556-6 34012113 160 Heller GT et al Targeting disordered proteins with small molecules using entropy Trends Biochem Sci 2015 40 9 491 496 10.1016/j.tibs.2015.07.004 26275458 161 Francois-Moutal L et al Direct targeting of TDP-43, from small molecules to biologics: the therapeutic landscape RSC Chem Biol 2021 2 4 1158 1166 10.1039/D1CB00110H 34458829 162 Pickhardt M et al Identification of small molecule inhibitors of tau aggregation by targeting monomeric tau as a potential therapeutic approach for tauopathies Curr Alzheimer Res 2015 12 9 814 828 10.2174/156720501209151019104951 26510979 163 Wang L et al Small molecule therapeutics for tauopathy in Alzheimer’s disease: walking on the path of most resistance Eur J Med Chem 2021 209 112915 10.1016/j.ejmech.2020.112915 33139110 164 Baggett DW Nath A The rational discovery of a tau aggregation inhibitor Biochemistry 2018 57 42 6099 6107 10.1021/acs.biochem.8b00581 30247897 165 Mirbaha H et al Inert and seed-competent tau monomers suggest structural origins of aggregation Elife 2018 7 e36584 10.7554/eLife.36584 29988016 166 Taniguchi S et al Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins J Biol Chem 2005 280 9 7614 7623 10.1074/jbc.M408714200 15611092 167 Akoury E et al Mechanistic basis of phenothiazine-driven inhibition of tau aggregation Angew Chem Int Ed Engl 2013 52 12 3511 3515 10.1002/anie.201208290 23401175 168 Schafer KN et al Structural determinants of tau aggregation inhibitor potency J Biol Chem 2013 288 45 32599 32611 10.1074/jbc.M113.503474 24072703 169 Mollasalehi N et al An allosteric modulator of RNA binding targeting the N-terminal domain of TDP-43 yields neuroprotective properties ACS Chem Biol 2020 15 11 2854 2859 10.1021/acschembio.0c00494 33044808 170 Abskharon R et al Cryo-EM structure of RNA-induced tau fibrils reveals a small C-terminal core that may nucleate fibril formation Proc Natl Acad Sci U S A 2022 119 15 e2119952119 10.1073/pnas.2119952119 35377792 171 Zhang H et al RNA controls PolyQ protein phase transitions Mol Cell 2015 60 2 220 230 10.1016/j.molcel.2015.09.017 26474065 172 Bhardwaj A et al Characterizing TDP-43 interaction with its RNA targets Nucleic Acids Res 2013 41 9 5062 5074 10.1093/nar/gkt189 23519609 173 Pozzi S et al Virus-mediated delivery of antibody targeting TAR DNA-binding protein-43 mitigates associated neuropathology J Clin Invest 2019 129 4 1581 1595 10.1172/JCI123931 30667370 174 Abskharon R et al Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer’s disease J Biol Chem 2020 295 31 10662 10676 10.1074/jbc.RA120.013638 32493775 175 Jicha GA et al Alz-50 and MC-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau J Neurosci Res 1997 48 2 128 132 10.1002/(SICI)1097-4547(19970415)48:2<128::AID-JNR5>3.0.CO;2-E 9130141 176 Nakamura K et al Proline isomer-specific antibodies reveal the early pathogenic tau conformation in Alzheimer’s disease Cell 2012 149 1 232 244 10.1016/j.cell.2012.02.016 22464332 177 Lu KP et al Potential of the antibody against cis-phosphorylated tau in the early diagnosis, treatment, and prevention of Alzheimer disease and brain injury JAMA Neurol 2016 73 11 1356 1362 10.1001/jamaneurol.2016.2027 27654282 178 Qiu C et al Cis P-tau underlies vascular contribution to cognitive impairment and dementia and can be effectively targeted by immunotherapy in mice Sci Transl Med 2021 13 596 eaaz7615 10.1126/scitranslmed.aaz7615 34078745 179 Kondo A et al Antibody against early driver of neurodegeneration cis P-tau blocks brain injury and tauopathy Nature 2015 523 7561 431 436 10.1038/nature14658 26176913 180 Albayram O et al Cis P-tau is induced in clinical and preclinical brain injury and contributes to post-injury sequelae Nat Commun 2017 8 1 1000 10.1038/s41467-017-01068-4 29042562 181 Porta S et al Patient-derived frontotemporal lobar degeneration brain extracts induce formation and spreading of TDP-43 pathology in vivo Nat Commun 2018 9 1 4220 10.1038/s41467-018-06548-9 30310141 182 Jucker M Walker LC Propagation and spread of pathogenic protein assemblies in neurodegenerative diseases Nat Neurosci 2018 21 10 1341 1349 10.1038/s41593-018-0238-6 30258241 183 Vaquer-Alicea J et al Tau strains shape disease Acta Neuropathol 2021 142 1 57 71 10.1007/s00401-021-02301-7 33830330 184 Koehler LC et al TDP-43 oligomerization and phase separation properties are necessary for autoregulation Front Neurosci 2022 16 818655 10.3389/fnins.2022.818655 35495061 185 Wagner J et al Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson’s disease Acta Neuropathol 2013 125 6 795 813 10.1007/s00401-013-1114-9 23604588 186 Ingham DJ et al Fungally derived isoquinoline demonstrates inducer-specific tau aggregation inhibition Biochemistry 2021 60 21 1658 1669 10.1021/acs.biochem.1c00111 34009955 187 Baggett DW Nath A Structure-activity relationships of novel tau ligands: passive fibril binders and active aggregation inhibitors ACS Chem Biol 2022 17 3 701 708 10.1021/acschembio.2c00012 35147406 188 Wagner J et al Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies Acta Neuropathol 2015 130 5 619 631 10.1007/s00401-015-1483-3 26439832 189 Seidler PM et al Structure-based discovery of small molecules that disaggregate Alzheimer’s disease tissue derived tau fibrils in vitro Nat Commun 2022 13 1 5451 10.1038/s41467-022-32951-4 36114178 190 Seidler PM et al Structure-based inhibitors of tau aggregation Nat Chem 2018 10 2 170 176 10.1038/nchem.2889 29359764 191 Owen I Shewmaker F The role of post-translational modifications in the phase transitions of intrinsically disordered proteins Int J Mol Sci 2019 20 21 5501 10.3390/ijms20215501 31694155 192 Jeon P et al Regulation of cellular ribonucleoprotein granules: from assembly to degradation via post-translational modification Cells 2022 11 13 2063 10.3390/cells11132063 35805146 193 Sternburg EL et al Post-translational modifications on RNA-binding proteins: accelerators, brakes, or passengers in neurodegeneration? Trends Biochem Sci 2022 47 1 6 22 10.1016/j.tibs.2021.07.004 34366183 194 Wesseling H et al Tau PTM profiles identify patient heterogeneity and stages of Alzheimer’s disease Cell 2020 183 6 1699 1713 10.1016/j.cell.2020.10.029 33188775 195 Farina S et al Post-translational modifications modulate proteinopathies of TDP-43, FUS and hnRNP-A/B in amyotrophic lateral sclerosis Front Mol Biosci 2021 8 693325 10.3389/fmolb.2021.693325 34291086 196 Rahman S. Posttranslational modifications of TDP-43. In: Kumar V, Jaiswal MK, eds. TDP-43 and Neurodegeneration. Elsevier; 2022:45–79. 197 Gruijs da Silva LA et al Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation EMBO J 2022 41 8 e108443 10.15252/embj.2021108443 35112738 198 Ukmar-Godec T et al Lysine/RNA-interactions drive and regulate biomolecular condensation Nat Commun 2019 10 1 2909 10.1038/s41467-019-10792-y 31266957 199 Cohen TJ et al The acetylation of tau inhibits its function and promotes pathological tau aggregation Nat Commun 2011 2 252 10.1038/ncomms1255 21427723 200 Yu H et al HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells Science 2021 371 6529 eabb4309 10.1126/science.abb4309 33335017 201 Shin MK et al Reducing acetylated tau is neuroprotective in brain injury Cell 2021 184 10 2715 2732 10.1016/j.cell.2021.03.032 33852912 202 Cohen TJ et al An acetylation switch controls TDP-43 function and aggregation propensity Nat Commun 2015 6 5845 10.1038/ncomms6845 25556531 203 Aikio M, et al. Opposing roles of p38α-mediated phosphorylation and arginine methylation in driving TDP-43 proteinopathy [preprint]. 10.1101/2021.08.04.455154 Posted on bioRxiv August 4, 2021 204 Stefanoska K et al Alzheimer’s disease: ablating single master site abolishes tau hyperphosphorylation Sci Adv 2022 8 27 eabl8809 10.1126/sciadv.abl8809 35857446 205 Riback JA et al Composition-dependent thermodynamics of intracellular phase separation Nature 2020 581 7807 209 214 10.1038/s41586-020-2256-2 32405004 206 Jiang L et al Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy Mol Cell 2021 81 20 4209 4227 10.1016/j.molcel.2021.07.038 34453888 207 Wheeler JM et al Activity of the poly(A) binding protein MSUT2 determines susceptibility to pathological tau in the mammalian brain Sci Transl Med 2019 11 523 eaao6545 10.1126/scitranslmed.aao6545 31852801 208 Apicco DJ et al Reducing the RNA binding protein TIA1 protects against tau-mediated neurodegeneration in vivo Nat Neurosci 2018 21 1 72 80 10.1038/s41593-017-0022-z 29273772 209 Becker LA et al Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice Nature 2017 544 7650 367 371 10.1038/nature22038 28405022 210 Vanderweyde T et al Interaction of tau with the RNA-binding protein TIA1 regulates tau pathophysiology and toxicity Cell Rep 2016 15 7 1455 1466 10.1016/j.celrep.2016.04.045 27160897 211 Ash PEA et al TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau Proc Natl Acad Sci U S A 2021 118 9 e2014188118 10.1073/pnas.2014188118 33619090 212 Lester E et al Tau aggregates are RNA-protein assemblies that mislocalize multiple nuclear speckle components Neuron 2021 109 10 1675 1691 10.1016/j.neuron.2021.03.026 33848474 213 Zuniga G et al Tau-induced deficits in nonsense-mediated mRNA decay contribute to neurodegeneration Alzheimers Dement 2023 19 2 405 420 10.1002/alz.12653 35416419 214 Elden AC et al Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS Nature 2010 466 7310 1069 1075 10.1038/nature09320 20740007 215 Zhang K et al The C9orf72 repeat expansion disrupts nucleocytoplasmic transport Nature 2015 525 7567 56 61 10.1038/nature14973 26308891 216 Bush JA et al Ribonuclease recruitment using a small molecule reduced c9ALS/FTD r(G4C2) repeat expansion in vitro and in vivo ALS models Sci Transl Med 2021 13 617 eabd5991 10.1126/scitranslmed.abd5991 34705518 217 Matsukawa K et al Long non-coding RNA NEAT1_1 ameliorates TDP-43 toxicity in in vivo models of TDP-43 proteinopathy RNA Biol 2021 18 11 1546 1554 10.1080/15476286.2020.1860580 33427561 218 Chatterjee B et al RNA modifications and RNA metabolism in neurological disease pathogenesis Int J Mol Sci 2021 22 21 10 10.3390/ijms222111870 34769301 219 Jiapaer Z et al Regulation and roles of RNA modifications in aging-related diseases Aging Cell 2022 21 7 e13657 10.1111/acel.13657 35718942 220 Roundtree IA et al Dynamic RNA modifications in gene expression regulation Cell 2017 169 7 1187 1200 10.1016/j.cell.2017.05.045 28622506 221 Drino A Schaefer MR RNAs, phase separation, and membrane-less organelles: are post-transcriptional modifications modulating organelle dynamics? Bioessays 2018 40 12 e1800085 10.1002/bies.201800085 30370622 222 Edupuganti RR et al N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis Nat Struct Mol Biol 2017 24 10 870 878 10.1038/nsmb.3462 28869609 223 Lee J-H et al Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation Mol Cell 2021 81 16 3368 3385 10.1016/j.molcel.2021.07.024 34375583 224 Zaccara S et al Reading, writing and erasing mRNA methylation Nat Rev Mol Cell Biol 2019 20 10 608 624 10.1038/s41580-019-0168-5 31520073 225 McMillan M et al RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia Mol Cell 2023 83 2 219 236 10.1016/j.molcel.2022.12.019 36634675 226 Kaushik S Cuervo AM Proteostasis and aging Nat Med 2015 21 12 1406 1415 10.1038/nm.4001 26646497 227 Alberti S Carra S Quality control of membraneless organelles J Mol Biol 2018 430 23 4711 4729 10.1016/j.jmb.2018.05.013 29758260 228 Labbadia J Morimoto RI The biology of proteostasis in aging and disease Annu Rev Biochem 2015 84 435 464 10.1146/annurev-biochem-060614-033955 25784053 229 Nixon RA The role of autophagy in neurodegenerative disease Nat Med 2013 19 8 983 997 10.1038/nm.3232 23921753 230 Vargas JNS et al The mechanisms and roles of selective autophagy in mammals Nat Rev Mol Cell Biol 2023 24 3 167 185 10.1038/s41580-022-00542-2 36302887 231 McMillan M et al RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia Mol Cell 2023 83 2 219 236 10.1016/j.molcel.2022.12.019 36634675 232 Yamasaki A et al Liquidity is a critical determinant for selective autophagy of protein condensates Mol Cell 2020 77 6 1163 1175 10.1016/j.molcel.2019.12.026 31995729 233 Jinwal UK et al Hsp70 ATPase modulators as therapeutics for Alzheimer’s and other neurodegenerative diseases Mol Cell Pharmacol 2010 2 2 43 46 20523917 234 Chiang AN et al Synthesis and evaluation of esterified Hsp70 agonists in cellular models of protein aggregation and folding Bioorg Med Chem 2019 27 1 79 91 10.1016/j.bmc.2018.11.011 30528127 235 Babu M et al Peptidyl prolyl isomerase a modulates the liquid-liquid phase separation of proline-rich IDPs J Am Chem Soc 2022 144 35 16157 16163 10.1021/jacs.2c07149 36018855 236 Hutten S et al Nuclear import receptors directly bind to arginine-rich dipeptide repeat proteins and suppress their pathological interactions Cell Rep 2020 33 12 108538 10.1016/j.celrep.2020.108538 33357437 237 Guo L et al Nuclear-import receptors reverse aberrant phase transitions of RNA-binding proteins with prion-like domains Cell 2018 173 3 677 692 10.1016/j.cell.2018.03.002 29677512 238 Caballero B et al Interplay of pathogenic forms of human tau with different autophagic pathways Aging Cell 2018 17 1 e12692 10.1111/acel.12692 29024336 239 Carrettiero DC et al Stress routes clients to the proteasome via a BAG2 ubiquitin-independent degradation condensate Nat Commun 2022 13 1 3074 10.1038/s41467-022-30751-4 35654899 240 Scotter EL et al Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species J Cell Sci 2014 127 pt 6 1263 1278 24424030 241 Yan Y et al X-linked ubiquitin-specific peptidase 11 increases tauopathy vulnerability in women Cell 2022 185 21 3913 3930 10.1016/j.cell.2022.09.002 36198316 242 Schaler AW et al PAC1 receptor-mediated clearance of tau in postsynaptic compartments attenuates tau pathology in mouse brain Sci Transl Med 2021 13 595 eaba7394 10.1126/scitranslmed.aba7394 34039738 243 Bourdenx M et al Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome Cell 2021 184 10 2696 2714 10.1016/j.cell.2021.03.048 33891876 244 Caballero B et al Acetylated tau inhibits chaperone-mediated autophagy and promotes tau pathology propagation in mice Nat Commun 2021 12 1 2238 10.1038/s41467-021-22501-9 33854069 245 Babinchak WM et al Small molecules as potent biphasic modulators of protein liquid-liquid phase separation Nat Commun 2020 11 1 5574 10.1038/s41467-020-19211-z 33149109 246 Fang MY et al Small-molecule modulation of TDP-43 recruitment to stress granules prevents persistent TDP-43 accumulation in ALS/FTD Neuron 2019 103 5 802 819 10.1016/j.neuron.2019.05.048 31272829 247 Kilgore HR Young RA Learning the chemical grammar of biomolecular condensates Nat Chem Biol 2022 18 12 1298 1306 10.1038/s41589-022-01046-y 35761089 248 Jonchhe S et al Small molecules modulate liquid-to-solid transitions in phase-separated tau condensates Angew Chem Int Ed 2022 61 23 e202113156 10.1002/anie.202113156 35320624 249 Dai B et al Myricetin slows liquid-liquid phase separation of tau and activates ATG5-dependent autophagy to suppress tau toxicity J Biol Chem 2021 297 4 101222 10.1016/j.jbc.2021.101222 34560101 250 Gray MJ et al Polyphosphate is a primordial chaperone Mol Cell 2014 53 5 689 699 10.1016/j.molcel.2014.01.012 24560923 251 Patel A et al ATP as a biological hydrotrope Science 2017 356 6339 753 756 10.1126/science.aaf6846 28522535 252 Zhang JZ et al Phase separation of a PKA regulatory subunit controls cAMP compartmentation and oncogenic signaling Cell 2020 182 6 1531 1544 10.1016/j.cell.2020.07.043 32846158 253 Peinado JR et al Sequestration of TDP-43216-414 aggregates by cytoplasmic expression of the proSAAS chaperone ACS Chem Neurosci 2022 13 11 1651 1665 10.1021/acschemneuro.2c00156 35549000 254 Alberti S Hyman AA Are aberrant phase transitions a driver of cellular aging? Bioessays 2016 38 10 959 968 10.1002/bies.201600042 27554449 255 Lashuel HA Rethinking protein aggregation and drug discovery in neurodegenerative diseases: why we need to embrace complexity? Curr Opin Chem Biol 2021 64 67 75 10.1016/j.cbpa.2021.05.006 34174698 256 Chen S et al Wolframin is a novel regulator of tau pathology and neurodegeneration Acta Neuropathol 2022 143 5 547 569 10.1007/s00401-022-02417-4 35389045