Protein misfolding and amyloid nucleation through liquid–liquid phase separation

Semanti Mukherjee ^a, Manisha Poudyal ^a, Kritika Dave ^b, Pradeep Kadu ^a and Samir K. Maji *^ab
aDepartment of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India. E-mail: samirmaji@iitb.ac.in
^bSunita Sanghi Centre of Aging and Neurodegenerative Diseases, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India

First published on 10th April 2024

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Semanti Mukherjee

Semanti Mukherjee is a PhD student at the Indian Institute of Technology Bombay (IITB) under the supervision of Prof. Samir K Maji. She obtained her MSc. in Chemistry from the Department of Chemistry (2016) at IITB. Her current research interests include liquid–liquid phase separation in functional and diseased amyloid.

Manisha Poudyal

Manisha Poudyal is an Institute Post-doctoral fellow in Prof. Samir K Maji's group at the Indian Institute of Technology Bombay. She holds a PhD in liquid–liquid phase separation of proteins and polypeptides under the supervision of Prof. Samir K Maji from IITB (2024). She obtained her MSc. degree in Biosciences and Bioinformatics from Tezpur University (2017). Her research is focused on the demonstration of multicomponent phase separation of proteins.

Kritika Dave

Kritika Dave is a project research assistant at Sunita Sanghi Centre of Aging and Neurodegenerative Diseases, Indian Institute of Technology Bombay, under the supervision of Prof. Samir K Maji. She obtained her master's degree in Neuroscience from King's College, London (2020). Her research area is focused on studying the liquid–liquid phase separation behaviour of proteins involved in neurodegenerative diseases.

Pradeep Kadu

Pradeep Kadu is a Post-doctoral fellow in Prof. Samir K Maji's group at the Indian Institute of Technology Bombay. He holds a PhD in Biophysical Chemistry and Nanomaterials under the supervision of Prof. Samir K Maji from the Indian Institute of Technology Bombay and co-supervision of Prof. Murali Sastry from Monash University, Australia (2023). He obtained his MSc degree in Chemistry from the Visvesvaraya National Institute of Technology, Nagpur, India. His research work is focused on understanding the origin of life, prebiotic conditions that may have led to the synthesis of peptides, and exploring the functional aspect of self-assembled peptides and proteins.

Samir K. Maji

Samir K. Maji is a professor at the Department of Biosciences and Bioengineering, IIT Bombay. He obtained his PhD from the Indian Association for the Cultivation of Science, Jadavpur, Kolkata, India, in 2003. He joined Harvard Medical School, USA, for his postdoctoral research in amyloid biology/biophysics and then moved to the University of California in Los Angeles, USA. He did his research associateship at Salk Institute, San Diego, USA, and ETH Zurich, Switzerland. Prof. Maji's work focuses on basic and applied areas of amyloid biology, including liquid–liquid phase separation and amyloid fibrillogenesis of proteins/peptides associated with human diseases and functions. His group also explores amyloid-based biomaterials for tissue engineering applications. He is currently professor-in-charge of Sunita Sanghi Centre of Aging and Neurodegenerative Diseases at IIT Bombay.

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1. Introduction

Neurodegenerative disorders refer to a wide variety of conditions characterized by the loss of a neuron's structure and functions due to glial and neuronal cell death.^1,2 These disorders have been observed as some of the intractable problems in the aging population worldwide.³ The three most common neurodegenerative disorders are Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS).⁴ Additionally, there are several other age-related chronic disorders, including multiple sclerosis (MS), frontotemporal dementia (FTD), Huntington's disease (HD), motor neuron diseases (MNDs), ataxia, multiple system atrophy (MSA), Creutzfeldt-Jakob disease (CJD) and various other dementias.^5,6 Individuals suffering from these diseases may experience problems with movement, speech, memory, cognition, intelligence, and more.⁷ As life expectancies in many nations continue to rise, the prevalence and incidence of these disorders are likely to increase in the foreseeable future. Several molecular investigations show that the aberrant amyloid aggregates of misfolded proteins serve as a common pathological hallmark in all neurodegenerative diseases and play a vital role in disease onset as well as its progression.^8–10 Amyloid fibrils are insoluble, highly ordered fibrillar aggregates, which are extremely stable under a wide variety of environmental conditions (Fig. 1a and b).^11,12 The analyses of purified fibrils using X-ray diffraction indicated that the constituent proteins are rich in a highly ordered, cross-β-pleated sheet structure with 4.7–4.8 Å intra-strand distance and 6–12 Å inter-sheet distance, which vary depending on the amino acid side chain composition and their packing in the cross-β-sheet arrangement (Fig. 1b).^13–15 Several unique properties such as tensile strength, insolubility in ionic detergents, and proteinase-K resistance are some distinct characteristics of amyloid that distinguish it from other disordered aggregates.^16,17 The majorly studied misfolded amyloids are from neuronal proteins, including hyperphosphorylated tau (p-tau),¹⁸ the amyloid-β protein (Aβ),¹⁹ α-syn,²⁰ the Huntingtin protein,²¹ and prion proteins,²² which are associated with an array of neurodegenerative diseases.²³ Moreover, misfolding of the stress granule-associated RNA-binding proteins such as FUS,²⁴ TDP-43,²⁵ TIA1,²⁶ and hnRNP family²⁷ also form pathogenic inclusions, which are associated with several neurodegenerative diseases such as ALS and FTD.^28,29 All these neurodegenerative diseases share some common features, such as the occurrence of brain lesions and an intracellular or extracellular accumulation of misfolded, ubiquitinated protein aggregates with amyloidogenic properties.¹⁰ For example, amyloid-β (Aβ) peptide accumulates as microscopic insoluble amyloid deposits, known as senile plaques, in AD.³⁰ Similarly, in PD, lesions are known as Lewy bodies, which are present in the cytoplasm and are predominantly composed of the protein α-syn.^31,32 Whereas in HD, lesions are observed as intranuclear and cytoplasmic inclusion bodies rich in Huntingtin protein.²¹ While the molecular pathology of FTD is still unknown, evidence suggests that FTD is caused by abnormal amyloid clusters, prevalently composed of TDP-43 and FUS, in frontal and temporal lobe neurons.^33,34 Recent research has also pointed out that these amyloid deposits are not solely responsible for the neurotoxic effects associated with these disorders.³⁵ Instead, soluble oligomers and protofibrils act as the proximate mediators of the diseases as they can easily transmit through the brain parenchyma and synaptic cleft affecting its structure and plasticity.^36,37 Indeed, oligomers of Aβ protein,³⁸ tau protein,³⁹ α-syn,⁴⁰ FUS,⁴¹ and Huntingtin⁴² are identified to play a crucial role in neurotoxicity as oligomers directly show concentration-dependent toxicity in neurons.⁴³ Thus, the current focus of the research in the amyloid field has shifted to understanding the early aggregation mechanism, which is not easy to achieve due to the heterogeneity of oligomers and their transient nature. Since the clinical regime for neurodegeneration mostly relies on symptomatic treatment, understanding the nucleation and early events of amyloid aggregation is particularly important for designing small molecules/therapeutics against protein aggregation. Recently, neurodegenerative diseases-associated proteins have been shown to undergo liquid–liquid phase separation (LLPS) where protein-rich condensates often undergo liquid-to-solid-like transition with the formation of amyloid-like assemblies.^44–46 Moreover, fibrillar species are shown to emerge out from the condensates upon aberrant amyloidogenic solidification suggesting that protein-rich condensates indeed have a direct role in the nucleation event for amyloid aggregation.^47,48 Interestingly, neurodegenerative disease-promoting factors such as familial mutations, gene multiplication, post-translational modification, metal ions exposure, oxidative stress, DNA damage, and the presence of RNA also facilitate phase separation events and subsequent viscoelastic liquid-to-solid transition.^49–55 Since the nucleation event is the rate-limiting step for amyloid aggregation,⁵⁶ it is reasonable to hypothesize that these factors may modulate LLPS and subsequent amyloid nucleation from condensates. Indeed, the amyloid aggregation pathway preceded by LLPS is emerging as a common aggregation mechanism for various neurodegenerative disease-associated proteins apart from the traditional pathway of amyloid aggregation from the bulk solution.^57–59 The present review focuses on the recent advancement in understanding amyloid aggregation from the aberrant phase separation. Specifically, the review addresses the molecular mechanism of amyloid nucleation and early-stage intermediate generation from the perspective of the emerging LLPS concept. The review also explains the underlying nucleation mechanism of protein self-assembly inside the liquid condensates, dissecting the molecular basis of viscoelastic liquid-to-solid transition, and proposes how metastable LLPS can serve as a misfolding/aggregation crucible for amyloid nucleation. Further, it also summarizes the effect of the common disease-associated factors on aberrant phase separation as well as subsequent amyloid aggregation. Overall, we propose that LLPS and the subsequent liquid-to-solid transition may serve as a generic mechanism for protein misfolding, aggregation and amyloid formation.

2. Protein misfolding and amyloid aggregation: a mechanistic overview

The molecular mechanism of protein misfolding and amyloid aggregation from the native state is not straightforward, as amyloid aggregation is not a simple two-step aggregation process. Instead, multiple higher-ordered “on” and “off-pathway” species, which are sometimes difficult to track due to metastability, are associated with the aggregation pathway.⁶⁰ Moreover, the same protein may follow multiple aggregation pathways depending on the genetic and environmental perturbation, protein concentration, and conformational plasticity.^61,62 Amyloid aggregation from all the proteins typically follows a nucleation-dependent polymerization mechanism,^63,64 when studied by the amyloid-specific thioflavin-T fluorescence enhancement, which exhibits sigmoidal growth kinetics with three consecutive steps: (1) a very slow nucleation or lag phase, (2) an exponential growth phase for fibril elongation, and (3) a stationary phase or dynamic equilibrium phase of all aggregated species with monomer (Fig. 1a and b).⁶⁵ The secondary structural transition of protein from the native state to the cross-β-sheet rich amyloid state can also be observed from circular dichroism spectroscopy, which is often used for the biophysical characterization of amyloids (Fig. 1b). The nucleation phase is the kinetic bottleneck in the whole aggregation process due to the high interfacial energy barrier, which prevents amyloid nucleation from the native monomeric protein.⁶⁶ However, once aggregation-competent nuclei are formed, the elongation phase is thermodynamically downhill with rapid monomer addition along with other secondary processes such as fragmentation, surface-catalyzed secondary nucleation, which produce oligomers, protofibril, and ordered fibrillar species (Fig. 1a).⁶⁷ It is important to note that the formation of aggregation-competent critical clusters is not a single-step event as explained by the classical nucleation theory.^68–70 Rather, amyloid nucleation typically follows a multistep non-classical nucleation pathway with minimal free energy change in each step instead of surpassing a high energy barrier in a single step.^71–73 Thus, the metastable pre-nucleation precursors, which further convert into stably growing nuclei, play an important role during the multistep nucleation event.⁷⁴ For example, folded proteins often undergo partial unfolding promoting non-specific intermolecular interaction to form micellar-like disordered oligomers, which serve as pre-nucleating clusters during amyloid nucleation.^71,75 Indeed, partially folded pre-molten globule-like conformation on the lag phase of several neurodegenerative disease-related proteins (such as α-syn, tau, Aβ, and TDP-43) is recognized as a key intermediate, which quickly initiates nucleation due to the solvent-exposed hydrophobic patches.^76–78 Interestingly, these partially folded states are reversible with the increase in the transient temperature but immediately induce oligomerization upon prolonged incubation at elevated temperature.⁷⁹

Moreover, the nucleation step can also be facilitated by the structural conversion of the early-stage oligomers into the self-catalyzing seeding-competent conformation, which promotes aggressive growth kinetics.⁸² Thus, the conformational conversion of oligomers may also act as a rate-limiting bottleneck in the whole aggregation process. For example, primarily disordered α-syn monomers get converted into helix-rich oligomers at the beginning of the elongation phase where the helix-rich conformation shows high aggregation propensity.⁸³ However, during the elongation stage, a dramatic increase in the β-sheet content is observed leading to proto-fibril and fibril formation. Oligomer formation upon protein misfolding indeed plays an important role in the nucleation event of other disease-associated proteins, such as Sup35 protein,⁸⁴ Aβ,^82,85 tau,⁸⁶ and Huntingtin protein.⁸⁷ The added complexity of the aggregation-competent structural conversion in the nucleation step is considered in the nucleation–conversion–polymerization model of amyloid aggregation, which indeed supports the multistep nature of amyloid nucleation.^84,88

Amyloid nucleation is the most crucial but poorly understood process due to the spatial and temporal resolution limit of instruments for detecting transient events. Primary nucleation is an important mechanism in the lag phase, which comprises the formation of the first aggregation-competent stable nuclei through monomer self-assembly (Fig. 2a).⁸⁹ When the primary nucleation event starts from a homogeneous protein solution, the process is known as homogeneous primary nucleation.⁹⁰ Since the monomer addition and dissolution rates are comparable in the homogeneous bulk solution, the primary nucleation becomes an exceedingly slow event. However, in the presence of a hydrophobic/hydrophilic interface or air–water interface, the primary nucleation kinetics often gets accelerated and the associated mechanism is termed heterogeneous primary nucleation (Fig. 2a).⁹¹ In the heterogeneous nucleation mechanism, the protein molecules attain a preferential conformation at the interface to maximize the overall hydrophilic and hydrophobic interactions, which promotes protein misfolding into nucleation-competent conformation due to surface adsorption.^82,92 Thus, most in vitro aggregation assays require sample agitation, which is often performed by shaking, stirring, or rotating protein to introduce an air–water interface for faster nucleation.⁹³ Moreover, nucleation-promoting surfaces, such as membranes,⁹⁴ phospholipid vesicles,⁹⁵ hydrophobic nanoparticles,⁹⁶ and beads⁹⁷ are also added externally to facilitate heterogeneous primary nucleation during the aggregation assays. If a protein follows only the primary nucleation mechanism, either it will never come out from the lag phase of aggregation or it will take an enormously long time to initiate the aggregation.⁶⁴ The lag phase gets shortened when secondary nucleation starts to take place where oligomers and preformed fibrillar species, known as seeds, act as templates for the self-catalytic addition of other oligomers and/or monomers (Fig. 2a).⁹⁸

Although fibril-catalyzed secondary nucleation and heterogeneous primary nucleation both follow a surface-mediated aggregation mechanism, the secondary nucleation process is much more aggressive due to multiple simultaneous processes such as fibril elongation, fragmentation, and autocatalytic aggregate formation (Fig. 2b), which are collectively exhibited as exponential growth kinetics of amyloid aggregation.^56,101 The primary nucleation step of the amyloid aggregation can be entirely surpassed by the dose-dependent addition of seeds.^89,102 Indeed the seeding nature of amyloid is observed for various neurodegenerative disease-associated proteins^99,100,103 and plays an important role in disease propagation in amyloidosis.⁶⁷ Interestingly, amyloid growth can follow distinct fibril amplification mechanisms depending on the seed size as experimentally observed in α-syn secondary nucleation. While the small seeds dominate the elongation mechanism through monomer addition, the large fibrillar seed facilitates surface-catalyzed secondary nucleation resulting in branching of the fibrils (Fig. 2c). Since fragmentation of fibrils can produce seeds with a range of size distribution, secondary nucleation is a complex heterogeneous process with variation in cellular uptake mechanism and cytotoxicity.

3. Oligomerization in the pathway of amyloid nucleation

Both the primary and secondary nucleation mechanisms result in the formation of early-stage oligomers, which play a crucial role in the multi-step nucleation mechanism of amyloid aggregation.^72,75 Oligomer represents a vast umbrella term comprising diverse populations of intermediate protein aggregates ranging from dimer to a few hundred-mers with various secondary structures, morphology, solubility, and stability.¹⁰⁴ Early-stage oligomers are metastable, highly soluble, and typically globular, annular, or linear in nature.¹⁰⁵ Importantly, recent research has established that early-stage soluble oligomers are the proximate mediator of neurotoxicity for most neurodegenerative diseases^{104,106–112} and exhibit direct concentration-dependent toxicity in neurons.^113,114 The toxicity of the early-stage, soluble oligomer arises due to their small size, high diffusivity, and surface hydrophobicity, which are responsible for an abnormal multitude of interactions with receptors, metabolites, nucleic acids, and cell membranes.^107,115 An important aspect of the secondary nucleation process is the associated neurotoxicity due to the aggressive production of heterogeneous oligomeric species.^116,117 Interestingly, some studies have pointed out that the ongoing process of amyloid aggregation with heterogeneous species populations shows more toxicity than the individual aggregated species.⁴³ For example, it has been shown that the surface-catalyzed secondary nucleation and the formation of oligomers of Aβ protein are directly proportional to cell death.¹¹⁸ In this line, the Lashuel group has proposed that Aβ toxicity is associated with the process of oligomers transitioning into the fibrillar state that triggers the intracellular cascade leading to cell death.¹¹⁹ On the other hand, Linse and co-authors proposed that fibrillation alone does not drive toxicity, rather, the generation of oligomers through fibril-assisted secondary nucleation is the most toxic event.⁶⁷ Indeed, the secondary nucleation mechanism has vast implications in neurotoxicity and is predominantly observed for many neurodegenerative disease-associated proteins, including Aβ,⁹⁹ tau,¹²⁰ α-syn,¹²¹ and prion protein.¹²² Although secondary nucleation aggressively generates toxic oligomers, small metastable oligomers can also be produced during the primary nucleation mechanism, which is enthalpically favored in the high monomer concentration.¹²³ Often, molecular crowder has been shown to increase amyloid aggregation by enhancing the local protein concentration due to the excluded volume effect.^90,124,125 In the diseased scenario, gene multiplication, compromised chaperone activity, and other disease-associated factors can elevate the local protein concentration above the physiological limit increasing the inherent threat of pathogenic oligomerization.^126,127 Another recently explored physiological condition, which concentrates protein molecules in a confined volume relies on the phenomenon of liquid–liquid phase separation (LLPS), which has been shown to play a huge role in protein self-assembly, oligomerization, and aggregation process.^46,128–131 The following section briefly describes the LLPS process, its potential role in the nucleation event of amyloid aggregation, and its implication in neurodegeneration based on the overwhelming number of recent publications.

4. Liquid–liquid phase separation (LLPS): an emerging field in biology

Cells, the fundamental building blocks of life, spatiotemporally control thousands of complex biochemical reactions through compartmentalization, segregating distinct chemical microenvironments within cellular space.¹³² Compartmentalization through membrane-bound organelles such as the nucleus, mitochondria, Golgi body and endoplasmic reticulum are well-understood through decades where membranes impede permeation of biomolecules as well as regulate molecular transport with specificity and selectivity.^133,134 However, cells also achieve compartmentalization through numerous membrane-less organelles such as nucleoli, Cajal bodies, PML bodies, paraspeckles in the nucleus; stress granules, Sec bodies, pyrenoid, germ granules, P Bodies in the cytoplasm; signaling clusters at the membrane (Fig. 3a).¹³⁵ Since these micron-sized assemblies lack any physical barrier for separating them from the cellular milieu, a long-standing curious research question is how these open macromolecular assemblies maintain their structure and functional integrity. It is interesting to note that back in 1899, Edmund B. Wilson imagined a cell as a densely packed colloidal system and hypothesized protoplasm as a mixture of liquids, in the form of a fine emulsion.¹³⁶ Moreover, in the early 1900s, Oparin and Haldane proposed that life originates within the membrane-less coacervate-like protocells from the Primordial Soup.¹³⁷ However, the concept of coacervation in cell physiology has recently gained attention when P granules from Caenorhabditis elegans embryos are reported as spherical liquid droplet-like assemblies formed through the process of the liquid–liquid phase separation (LLPS).¹³⁸ Over the last decade, LLPS has emerged as a wide-ranging generic phenomenon for membrane-deprived compartmentalization of biomolecules (proteins and nucleic acids), collectively referred to as “biomolecular condensates” due to their condensing property in the cellular milieu (Fig. 3a).^139–142 The liquid-like condensates exhibit fast molecular diffusion, which can be tracked by the fluorescence recovery after the photobleaching (FRAP) experiment (Fig. 3b). Other classical features include droplet fusion, thermo-reversibility, and surface-wetting properties that are typically observed in a wide range of biomolecular condensates (Fig. 3c–e). These condensates spatiotemporally regulate a myriad of cellular functions including reversible stress response,¹⁴³ cell signaling,¹⁴⁴ vesicular trafficking,¹⁴⁵ transcriptional regulation,¹⁴⁶ genomic organization,¹⁴⁷ molecular sequestration,^148,149 and even cellular fitness.¹⁴³ Interestingly, the phase-separated condensates can also serve as the reaction crucible for physiologically relevant nucleation reactions, which are observed in the case of mitotic spindle¹⁵⁰ and heterochromatin formation.¹⁵¹ Mitotic spindles are majorly nucleated inside membrane-deprived organelles, named centrosomes.¹⁵² The spindle formation is initiated through microtubule polymerization where locally concentrated tubulin nucleates the polymerization reaction.¹⁵³ Moreover, functional phase-separation of the microtubule-associated protein tau has been shown to drive the nucleation process of microtubule formation.¹⁵⁴ Similarly, heterochromatin formation initiates from the formation of the nucleated foci from HP1α protein through the process of LLPS.¹⁵⁵

However, the physiological nucleation through biomolecular condensates is actively regulated by cellular homeostasis to prevent the inherent risk of aberrant nucleation of biomolecular aggregation. The intermolecular interaction strength of biomolecules is a key regulatory factor for physiological LLPS, which is often modulated by appropriate environmental conditions (such as pH, salt, and biomolecule concentration) in healthy cells.^161,162

The primary driving force for biological LLPS is intermolecular interaction, which can be quantitatively evaluated based on Flory Huggins's theory of polymers.¹⁶³ In this model, a polymer solution is considered as an infinite lattice where each lattice point is filled by either a polymer or a solvent molecule. So, in the lattice, three types of intermolecular interactions are possible. They are polymer–polymer, polymer–solvent, and solvent–solvent interaction. The empirical Flory interaction parameter (χ) measures the strength of intermolecular interaction, predicting the molecular arrangement in the lattice to minimize the free energy of the system. A negative value of χ (χ < 0) indicates a “good solvent” where heterotypic protein–solvent interactions dominate over homotypic interactions inhibiting phase separation. However, if the polymer–polymer interaction energy is very high in the scale of thermal energy, χ value will be positive (χ > 0), indicating a “poor solvent” condition, which favors phase separation of a polymeric system. Protein indeed can be considered as an associative biopolymer consisting of an array of amino acids where peptide dipole from polar amino acids gives rise to a net attraction between polypeptide backbones promoting phase separation.^164–167 While Flory Huggins's theory oversimplifies the polymer nature considering them as a homopolymer; protein, on the other hand, is a complex heteropolymer with diverse amino acid sequences that give rise to multiple simultaneously acting unique intermolecular interactions.¹⁶⁸ The molecular feature of protein and polypeptide driving LLPS is encoded in the primary amino acid sequence, where the cumulative effect of weak non-specific multivalent interactions such as ionic, hydrophobic, hydrogen bonding, cation–π interactions, and π–π interactions have been shown to dictate LLPS propensity^169–173 (Fig. 3f). Multivalency is the governing force for phase separation, arising from different local structures of both folded and disordered proteins.¹⁴¹ Well-folded globular domains with predisposed associative interaction surfaces can act as “patchy colloids” driving phase separation.^174–176 Often multiple repetitive folded domains (e.g. SH3 domains) interspersed by flexible linkers undergo phase separation through multivalency, which has been profoundly observed in signaling clusters.¹⁴⁵ The recently developed sticker-spacer model of protein phase separation describes how multivalency arises from the combination of folded and flexible domains.^177,178 However, the predominant contributor of multivalency comes from the intrinsically disordered region (IDR) of the proteins with conformational flexibility, which samples multiple weak non-specific interactions simultaneously.^179,180 The IDR sequences are often enriched in charged residues (such as aspartic acid, glutamic acid, lysine, and arginine), aromatic residues (such as phenylalanine and tyrosine), and polar residues (such as serine, proline, and glutamine) resulting low-complexity domains (LCD) in disordered proteins. The LCD bias in protein sequence encodes the “fuzzy” interaction mode mediating a multitude of simultaneously showing multivalent interactions.^181–183 On the other hand, prion-like domains are another specific class of IDR that is largely devoid of charged residues but enriched with polar moieties (such as serine, tyrosine, glutamine, and asparagine) and aromatic residues (such as phenylalanine and tyrosine) driving phase separation through weak, nonspecific cation–π and π–π interactions interaction.¹⁸⁴ Apart from the multivalency, the specific patterning of amino acids in the polypeptide sequence such as a pair of high oppositely charged residues, also promotes intermolecular interaction and phase separation.¹⁷¹ Thus, it is essential to note that instead of a specific intermolecular interaction, different types of interactions based on protein sequence and their pattering contribute simultaneously to generate a hierarchical interplay of intermolecular interactions that results in LLPS of proteins and polypeptides.^{170,179,185,186} Interestingly, IDR, LCD, and PrLD, which are the predominant driving factors for LLPS are profoundly present in all neurodegeneration-disease-related proteins, making them suitable for undergoing LLPS.^187–189 For example, TDP-43 and FUS contain large stretches of PrLD;¹⁹⁰ whereas α-syn and tau are intrinsically disordered in nature with LCDs containing charged residues.¹⁹¹ Indeed, recent studies have shown that major neurodegenerative disease-associated proteins such as α-syn,⁴⁷ tau,¹⁶⁰ Prion protein,¹⁹² Huntingtin,¹⁹³ FUS,¹⁹⁴ TDP-43,⁴⁸ and hnRNPA1,¹⁹⁵ TIA1,¹⁹⁶ C9orf72¹⁹⁷ have an inherent property to undergo phase separation. However, the nature of the major intermolecular interaction driving LLPS of neurodegeneration-associated proteins may vary from protein to protein. While π–π and cation–π interactions in the LCD drive LLPS of FUS, TDP-43, and hnRNPA1,^48,195,198 whereas tau protein undergoes LLPS mainly through electrostatic interaction;^160,199 α-syn LLPS is mainly driven by the hydrophobic interaction from the exposed NAC domain.¹⁵⁸ When these aggregation-prone proteins accumulate inside liquid droplets, the local concentration of the protein becomes enormously high and raises an innate threat for aggregation. Thus, understanding the time-dependent changes within biomolecular condensates and their consequence in protein aggregation has become a progressively important research aspect.

5. Liquid-to-solid phase transition: implication in neurodegeneration

Most intracellular functional condensates are liquid-like and highly dynamic in nature with rapid molecular exchange and environment-responsive reversibility, which are critical for their functional purpose.²⁰⁰ The liquid-like dynamicity of condensates arises from short-range, weak, nonspecific interaction, which rapidly breaks and reforms within a pico-to nanosecond timescale.²⁰⁰ Moreover, the biological liquid is inherently active with persistent energy-consuming reactions and molecular flux, which maintains its extremely dynamic nature, quick reversibility, and molecular composition.^142,201 In this regard, it is important to note that biomolecular condensate liquids are not simple Newtonian liquids (whose viscosity is independent of the rate of sheer stress).²⁰² Rather, they behave as viscoelastic Maxwell fluid with a complex network-like arrangement where network strength dictates different material states of biomolecular condensates.^203,204 Hence, biomolecular condensates are extremely metastable, where the protein-dense liquid phase can be transitioned into a range of solid-like condensed phases (such as gel, amorphous aggregates, glassy solid, kinetically-trapped assemblies, crystal, and amyloid fibrils) (Fig. 4). This solid-like transition is mainly assisted by strong long-range interactions that provide a long bond lifetime, structural specificity, less dynamicity, and more stability.²⁰⁵ The solid-like condensates serve important functionalities in cells; such as molecular sequestration (e.g., Balbiani Body, Amyloid-Bodies), organization hub (e.g., heterochromatin), reaction crucible (e.g., centrosomes), permeability (e.g., nucleopore complex), stress adaptation (e.g., stress granule), and polymerization (e.g., microtubule) (Fig. 4a). However, the active cellular machinery tightly regulates the condensate's material property, preventing the time-dependent aberrant solidification. Moreover, the active cellular processes also regulate the time-dependent disassembly of solid-like, non-dynamic condensates after serving their functionality.¹⁴⁹ For instance, time-dependent viscoelastic transition has been observed as a stress survival strategy for yeast prion protein SUP35, which undergoes reversible gelation upon molecular sequestration (Fig. 4b).²⁰⁷ Even, functional amyloids formed through the liquid-to-solid transition of biomolecular condensates, such as Balbiani bodies and amyloid bodies, also undergo time-dependent dissolution after their functions, suggesting that cells indeed strategically utilize the diverse material states of condensates for their benefits (Fig. 4c).¹⁴⁹ However, in the compromised cellular machinery, the unregulated viscoelastic transition is often irreversible in nature, which can be deleterious to cells.^46,210

In fact, several human diseases, ranging from genetic disease,²¹¹ metabolic diseases,²¹² cardiovascular disease,²¹³ viral infection,^214,215 neurodegeneration,^{44,45,216–218} and cancer^219,220 are reported to be preceded by aberrant liquid-to-solid phase transition of biomolecular condensates. During the aberrant liquid-to-solid transition, the physical cross-linking in the network-like liquid can enhance with time leading to vitrification or kinetically arrested states (e.g. gel or amorphous glassy solids) (Fig. 4a).²²¹ It was suggested that the complex network-like microenvironment arises from the sequence-specific multivalent aromatic interaction, prevalently present in PrLD, which acts as stickers while spacer amino acids (such as glycine, serine, and glutamine) impart flexibility, reversibility, solubility and regulate the overall liquid-like material property of condensates.¹⁶⁹ Above a threshold sticker concentration in protein, a system-spanning geometric transition exists, resulting in percolation or gelation due to multiple associative intermolecular interactions from sticker residues.²²² Apart from the physical cross-linking, a second type of liquid-to-solid transition of protein condensates may occur due to the entanglement of polymeric chains around each other, resulting in amorphous solidification (Fig. 4a).^223,224 A third type of liquid-to-solid transition can occur when the high local protein concentration within condensates either allowing the otherwise forbidden molecular interactions or triggering protein misfolding, resulting in the most stable cross-β-sheet rich amyloid aggregation (Fig. 4a).²³⁰ Indeed, several neurodegeneration disease-related protein condensates often lose the liquid-like dynamic properties (e.g., diffusion, coalescence, reversibility, etc.) with time and aberrantly produce amyloidogenic fibrous aggregates (Fig. 5).^{43,210,232,233} For example, transmission electron microscopy images clearly showed time-dependent morphology change of α-syn condensates where fibrils burst out from aged condensates (Fig. 5).⁴⁷ Similarly, RNA-binding proteins FUS and hnRNPA1, also show liquid-like dynamic condensates at early time points, which rigidify with time (Fig. 5).^195,227 Moreover, fibrils have been seen to protrude from the surface of the solid-like FUS condensates finally adopting a start-burst-like fibrous morphology upon condensate aging (Fig. 5).¹⁹⁴ These solid-like condensates of neurodegenerative proteins, such as α-syn, tau, FUS, TDP-43, and Prion protein also bind to amyloid-specific dyes, like thioflavin-S and thioflavin-T, thus confirming their amyloid-like nature.^47,160,234 Often the aberrant liquid-to-solid transition of the condensates from these neurodegenerative disease-related proteins results in irreversible mesh-like hydrogel formation, comprising amyloid aggregates, which has been reported for α-syn,⁴⁷ tau,²³⁵ FUS,¹⁹⁸ TDP-43,²³⁶ hnRNPA1,²³⁷ hnRNPA2.²³⁸ These porous hydrogel-entrapping solid condensates may act as a repository for toxic oligomers and other aggregated species. The hypothesis was strengthened based on our previous observation that α-syn hydrogels entrap helix-rich oligomers and short fibrils and exert neurotoxicity.²³⁹ Short-repeat-containing RNA, which is hugely deposited in the pathogenic inclusions such as in the case of HD, ALS, FTD, and muscular dystrophy, also plays a critical role in aberrant phase transition and sol–gel transition of these protein condensates.²⁴⁰ Consistent with the in vitro LLPS, phase separation of proteins associated with neurodegeneration is extensively observed in the relevant cellular microenvironment, which is converted into pathogenic inclusions by following various mechanisms.^{47,160,195,227,231} For example, disease-relevant conditions promote aberrant condensate formation leading to rapid transitions into pathogenic solid-like condensates (Fig. 6a).^47,241

On the other hand, solid-like stress granule condensates, which were initially functional, can be converted into pathogenic solid-like condensates upon prolonged stress exposure²⁴² (Fig. 6a). Indeed, all the major proteins associated with neurodegeneration, such as α-syn,⁴⁷ tau,¹⁶⁰ FUS,²²⁷ TDP-43,²³¹ Huntingtin,²³² and hnRNPA1¹⁹⁵ have been experimentally observed to form aberrant condensates in a cellular milieu with potential pathological implications (Fig. 6b). For example, α-syn forms liquid-like pan cellular condensates in HeLa cell upon metal ion stress, which gradually covert into amyloidogenic solid assemblies and accumulates in the perinuclear region forming aggresome (Fig. 6c).⁴⁷ The viscoelastic liquid-to-solid transition of α-syn condensates was also observed in the in vivo C. elegans model where the aged C. elegans accumulates amyloid-like inclusions, enriched with ubiquitin (Fig. 6d).²³³ The growing evidence on amyloid-like condensate maturation preceded by LLPS suggests that LLPS may play a key role as an early molecular event of pathogenic protein aggregation associated with neurodegeneration.

6. Molecular mechanism of amyloid aggregation from biomolecular condensates

6.1. Nucleation mechanism associated with aberrant phase separation

Amyloid aggregation from protein monomers follows an interconnected multistep nucleation process, which dictates the complexity of the nucleation landscape.²⁴³ The phenomenon of LLPS can potentially serve as an intermediate step where the nucleation and growth of biomolecular condensates make a suitable microenvironment to facilitate the nucleation toward amyloid aggregation further. Thus, to elucidate the molecular mechanism of amyloid aggregation from biomolecular condensates, one needs to focus on two overlapping nucleation events; the nucleation mechanism for LLPS along with the nucleation mechanism for liquid-to-amyloid transition. The biological phase separation may be initiated through a nucleation-dependent growth mechanism where intermolecular interaction dictates the formation of growth-competent critical nuclei above a threshold solute concentration, designated as the saturation concentration.²⁴⁴ Above the saturation concentration, the protein solution enters the metastable binodal regime, where thermal fluctuation initiates a nucleation event for phase separation. The nucleation event for physiological phase separation is an active process in the cellular microenvironment that regulates the formation, growth and dissolution of the biomolecular condensates.^140,245 In this context, a recent study quantitatively investigated early molecular events of α-syn and Synphilin1 protein (α-syn interacting protein and a common tracer for the misfolded protein aggregates in LB), in the growth conditions of healthy mammalian cells.²⁴⁶ The study indicated that healthy mammalian cells contain heterogeneous size distribution of protein clusters, indicating a supersaturated protein solution associated with a nucleation energy barrier where clusters above a critical size are capable of further growth. Interestingly, the putative chaperone maintains tight proteostasis regulation, which preferentially disassembles large clusters, thus avoiding surpassing the nucleation energy barrier toward aberrant cluster growth.²⁴⁶ Important to note that, the nucleation and coalescence kinetics of biomolecular condensates of physiologically regulated phase separation are distinctly different from those of unregulated pathological phase separation, which shows altered size-distribution of condensates as well as their abnormal growth dynamics.²⁴⁷ Actively regulated native condensates show exponential size distribution with a single mean size as a result of discrete nucleation events whereas neurodegeneration-associated mutant Huntingtin protein yields broad power-law size distribution of condensates as a result of continuous nucleation events.²⁴⁷ Transient oligomerization of protein is also an important regulatory factor in modulating the nucleation energy barrier for LLPS.^248–252 For example, TDP-43 can undergo phase separation without low complexity PrLD but the N-terminal oligomerization domain is essential for phase separation.²⁵³ Mutation and other structural modifications in the oligomerization domain can potentially alter the nucleation energy barrier of the phase separation with pathological implications. For example, UBQLN2, a protein quality control member of stress granule, undergoes LLPS through its proline-rich oligomerization domain.²⁵⁴ Interestingly, ALS-associated mutation on the proline-rich domain of UBQLN2 enhances its oligomerization propensity, decreasing the saturation concentration of LLPS and promoting an aberrant liquid-to-solid transition.²⁵⁵ Oligomer size can also vary with disease-associated mutation, as seen in tumor suppressor speckle-type POZ protein (SPOP), which is capable of altering the critical cluster formation rate, in other words, the nucleation rate.²⁵⁶ Transient oligomeric self-assembly may form nanoclusters below the saturation concentration, playing an important role in nucleating macroscopic phase separation, which indeed has been experimentally detected for several neurodegeneration-related proteins.^{249–252,257} For example, micron-sized condensate formation of hnRNPA1 low complexity domain follows a multi-step nucleation mechanism, which starts by forming low-affinity nanoclusters at the sub-saturated protein concentration.²⁵⁰ After the initial energetically uphill nanocluster formation, the monomer starts to be recruited with high affinity to form the observable condensates. A similar nanoscale cluster formation in the subsaturated protein concentration is observed for other disease-associated proteins, such as FUS, α-syn, and other RNA-binding proteins.^249,252 As the protein concentration increases from sub-saturation concentration to saturation concentration, sequence-encoded molecular interaction drives nanoclusters toward macroscopic phase separation.²⁵² Mutations, post-translational modification, and other molecular interactions can significantly alter the kinetics of these early molecular events, directly impacting the nucleation rate for aberrant protein aggregation.^249,252 Indeed, Alzheimer's disease-associated familial mutations P301L and P301S of tau protein are shown to enhance the kinetics of pathogenic amyloid fibrillation from LLPS, initiated through nanocluster formation at subsaturated concentration.²⁵⁷ However, cluster formation at subsaturated concentration and macroscopic phase separation at supersaturated concentration may not always be coupled with each other as several mesoscopic clusters have been reported and many of them directly nucleate amyloid fibrils without forming macroscopic phase separation.^258–261 Future research is necessary to explore the role of different mesoscale clusters in the complicated nucleation mechanism of LLPS. The regulation in the nucleation process of LLPS is indeed the key factor for preventing aberrant phase separation. The cellular regulation is even more challenging because most proteins are expressed close to their solubility limit as indicated by a proteome-wide study on Caenorhabditis elegans.²⁶² This is advantageous for nucleating functional LLPS but alarming for cells against pathological nucleation. Moreover, the same study also showed that during aging, the total quantity of protein aggregation sharply increases and cellular machinery for maintaining protein solubility is gradually compromised.²⁶² Since cellular protein concentration is on the verge of solubility, the nucleation event will likely act as a checkpoint for preventing aberrant phase separation in cells by active regulation.

6.2. Amyloid nucleation from biomolecular condensates

Amyloid aggregates are the most stable thermodynamic states in the energy landscape of protein aggregation while biomolecular condensates are inherently metastable, especially for aggregation-prone proteins.²⁶³ The enhanced intermolecular interaction and reduced desolvation energy upon LLPS energetically favor the primary nucleation of amyloid aggregation, which is otherwise an extremely slow bottleneck process in the traditional aggregation pathway.²⁶⁴ Although the precise molecular mechanism of primary nucleation is not yet fully understood, it is reasonable to speculate that the high local protein concentration within the condensate can alter the native intermolecular interaction of protein, facilitating protein misfolding. Intriguingly, most of the neurodegeneration-associated intrinsically disordered proteins upon phase separation show conformational bias towards extended conformation due to stabilizing lateral intra-chain contacts, which might allow the otherwise inaccessible critical interaction for initiating amyloid nucleation.^{158,257,265–272} For example, the autoinhibitory conformation of α-syn due to the electrostatic interaction between the N-terminal and C-terminal protects the NAC domain from surface exposure.¹⁵⁸ However, low pH or high salt conditions screen the surface charge of α-syn, disrupting the autoinhibitory conformation and leading to the exposure of the hydrophobic NAC domain.²⁶⁹ The alteration of intermolecular interaction due to conformational change is likely to reduce the nucleation barrier, manifesting as a faster liquid-to-amyloid transition at low protein concentration. Similarly, the autoinhibitory “paperclip” conformation of tau also gets extended upon LLPS exposing the microtubule-binding domain (MTBD).^257,268 The aggregation-prone hexapeptide and regulatory KXGS motifs in the exposed MTBD domain attain a transient β-hairpin structure in the phase-separated condition leading to irreversible amyloid aggregation through phase separation.²⁷³ Full-length prion protein also undergoes conformational expansion during phase separation where exposed N-terminal proline and glycine-rich octapeptide (PHGGGWGQ) repeat rapidly to form a short cross-β motif, which drives amyloid aggregation within condensates.²⁷¹ Protein misfolding due to conformational expansion can also be triggered at the condensate interface to maximize the protein–protein and protein–solvent interaction simultaneously.^{225,228,274,275} For example, the prion-like low complexity domain of protein hnRNPA1 shows reversible percolated crosslinking architecture in the condensate's interior while the interface shows the most-expanded protein conformation with a preferential perpendicular orientation toward the interface.²⁷⁰ Interestingly, the condensate interface possesses some unique molecular properties compared to the bulk interior, which may lead to a faster solidification.²⁷⁰ Indeed, the liquid-to-gel transition of FUS condensates is also experimentally observed to initiate from the interface, reminiscing a core–shell inhomogeneous architecture, where the fibrillar network gradually propagates towards the center.²²⁵ Moreover, amyloid fibrillation can preferentially occur only from the condensate interface, not from the bulk solution as observed for condensates of the low-complexity domain of hnRNPA1.²²⁸ Similarly, super-resolution single-molecule spectroscopy on FUS condensate revealed the formation of distinct nanoscale amyloid aggregates, which preferentially form on the condensate surface and restrict the local molecular diffusion generating a heterogeneous architecture of low-diffusive shell and diffusion-permitting condensate interior.²⁷⁵ Thus, the spatiotemporal heterogeneous microenvironment of condensate can effectively nucleate amyloid fibrillation, representing an emerging novel aggregation mechanism for disease-associated proteins.

6.3. Oligomer toxicity and polymorphism associated with LLPS-mediated aggregation pathway

The conventional amyloid aggregation process often generates non-fibrillar off-pathway oligomers and pre-fibrillar on-pathway oligomeric species, which exert proximate cytotoxicity as compared to the matured amyloid fibrils.^276–279 In fact, in some cases, the formation of the inclusion bodies has been proposed to be a neuroprotective response for sequestrating toxic oligomers and aggregated species.²⁸⁰ Interestingly, on-pathway and off-pathway toxic oligomers are also observed to accumulate within condensates, deciphering their pathological relevance in neurodegeneration. For example, prolonged incubation of tau condensate results in a time-dependent accumulation of soluble neurotoxic oligomers, which is further accelerated by FTD-associated mutation P301L.²⁶⁸ These oligomer-rich tau condensate does not significantly populate fibrillar aggregates with time suggesting that phase separation could be a mechanism to accumulate toxic “off-pathway” oligomers. Indeed, stress-granule protein TIA1 (T-cell intercellular antigen 1), which is a known driving factor for tau oligomerization and tau-dependent neurotoxicity, promotes the formation of toxic oligomers upon phase separation and inhibits the conversion of oligemic tau into fibrillar aggregates.¹²⁹ Interestingly, small molecule-based inhibitors, such as C1 and Shikonin are designed to target LLPS-mediated oligomerization to alleviate cytotoxicity in neuroblastoma cells.^281,282 A crucial factor in the toxicity mechanism is the membrane interaction where misfolded oligomers interact with membrane protein and disrupt biological membranes leading to mitochondrial dysfunction, calcium imbalance and reactive oxygen species generation.²⁸³ For example, soluble amyloid-β oligomers (AβOs) are known to trigger downstream neurotoxicity by binding cell surface receptors.²⁸⁴ A recent study showed that membrane-mimicking in vitro conditions can induce LLPS of AβOs where the protein attains membrane-binding toxic α-helical conformation.²⁸⁵ Prolonged incubation of AβOs condensates leads to amyloid fibril formation suggesting a possible on-pathway toxic oligomerization during LLPS. In this regard, another study has reported that amyloid-β oligomers isolated from AD autopsy brain lysate can co-phase separate with cellular prion protein at the plasma membrane leading to toxic hydrogel formation, which can trap neurotoxic co-receptor mGluR5.²⁸⁶ Similar to amyloid-β oligomers, our lab has previously observed that the structural transition of α-syn into cross-β amyloid accelerates through the formation of hydrophobic surface exposed helix-rich oligomers, which show cytotoxicity to SHSY-5Y cells.⁸³ These cytotoxic α-helical oligomers have also been shown to accumulate in the hydrogel formed through the cross-β fibrillation.²³⁹ Interestingly, α-syn phase separation also generates a certain population of helix-rich oligomers within the condensates during the time course of condensate aging indicating a potential link with oligomer toxicity in the novel aggregation pathway.⁴⁷ Indeed, a recent study has shown that a class of evolutionary conserved small EDRK-rich factors (SERF), which impede the accumulation of α-synuclein oligomers during the LLPS-mediated amyloid aggregation, exhibit reduced cellular toxicity.²⁸⁷ Thus, SERF might accelerate the conversion of highly toxic oligomers to less toxic amyloid aggregates to mitigate oligomer toxicity as a protective mechanism. Often, disease-associated mutation disrupts the physiological oligomerization and phase separation resulting not only in the loss of native functional role but also in gaining toxicity. For instance, TDP-43 self-oligomerization is a key step to trigger functional phase separation, which is hampered by ALS-associated mutations, A315T and M337V.^288,289 These mutations not only hinder the native autoregulatory function of condensates but also disrupt the liquid property of condensates resulting in compromised protein homeostasis. Similarly, FTD-associated mutations (ΔK280, P301L, P301S, A152T) trigger the pathogenic oligomerization of tau during LLPS, which acts as a precursor for amyloid aggregation.¹⁶⁰ However, further investigation is necessary to explore the mechanism of oligomerization from condensates and their structure–toxicity relationship in disease progression. Future research should also thoroughly investigate the role of membrane interaction in the toxicity mechanism of phase-separated oligomer-rich assemblies. Polymorphism is another classical feature of amyloid fibril at the molecular level, which generates multiple distinct fibrillar strains from the same protein with strain-specific diverse biological activity.^81,290–293 The polymorphic nature of amyloid fibril results in clinical and pathological variation, which further complicates the toxicity mechanism in neurodegeneration.^81,294,295 Interestingly, the LLPS-mediated aggregation pathway can produce distinct fibrillar polymorphs, as recently observed for α-syn.²⁹⁶ α-Syn aggregates preferentially attain an antiparallel β-sheet arrangement when initiated from a water-poor droplet microenvironment in contrast to the parallel β-sheet conformer commonly found when preceded at hydrophilic/hydrophobic interface.²⁹⁶ Interestingly, toxic oligomers of α-syn have been previously reported to generate aggregated species with antiparallel β-sheet conformation, which was speculated as “off-pathway” intermediates.^297–299 The novel LLPS-mediated aggregation pathway may explain these intermediates as “on pathway” species and may also indicate a strong connection with toxicity. It is important to note that, high-resolution structural studies revealed that the in vitro amyloid fibrillar strain differs significantly from the patient-derived strain, as reported for several neurodegeneration-associated proteins.^300–302 Future research should explore the high-resolution structural details of amyloids formed from the crowded protein-rich condensates, which may elucidate a new insight into fibril polymorphism and its implication in in vivo pathogenicity.

6.4. Seeding-dependent amyloid aggregation within biomolecular condensates

Amyloid aggregation within biomolecular condensates can also follow autocatalytic amplification and seeded kinetics, which are the classical mechanisms for aggressive fibril production (Fig. 7a). Since liquid-like nascent condensates allow rapid molecular exchange, small preformed fibrillar seeds can be internalized rapidly within the condensates and can act as a template to initiate aberrant aggregation. Indeed, the addition of pre-formed fibrillar seed triggers dose-dependent rapid amyloid aggregation from α-syn condensates suggesting seeded-kinetics as a prominent molecular event in the novel aggregation pathway (Fig. 7b and c).³⁰³ Moreover, when exposed to preformed fibrillar seeds, α-syn condensates formed in HeLa cells and PD-relevant SH-SY5Y cells undergo a liquid-to-solid transition into phosphorylated amyloid aggregates, which may aid in prion-like cell-to-cell-transmission (Fig. 7d).³⁰⁴ Apart from the external addition of seeds, the amyloidogenic aggregates produced within condensates can also serve the seeding purpose for further auto-amplification of aggregates (Fig. 7a). For example, the liquid-to-solid transition of human prion protein shows the seeding behaviour producing self-replicating autocatalytic amyloid aggregates, a characteristic of the prion-like transmission mechanism.²²⁹ Sometimes, a small segment of a few amino acids within a large protein may have a high propensity for cross-β sheet structure formation, which may also act as an efficient endogenous seed in the concentrated microenvironment of phase separation to trigger the overall protein aggregation into amyloid. The phenomenon has been observed for the FUS low complexity domain where short amyloid-prone peptide segments show seeded aggregation kinetics on full-length FUS through a phase separation.³⁰⁵ Interestingly, in the phase-separated system, the presence of tiny seeds that are either endogenously formed (or added exogenously), can initiate simultaneous amyloid aggregation in the dense and dilute phases. This might lead to polymorphic amyloid formation from the same solution (Fig. 7a). For instance, the liquid-to-solid transition of TDP-43 condensates results in the accumulation of β-sheet-rich amyloid fibrillar structure within condensates. Interestingly, amyloid aggregation outside the condensate is also observed at a later time point indicating multiple simultaneously acting amyloid aggregation mechanisms in the LLPS solution.³⁰⁶ Intriguingly, the study further revealed that the amyloid fibrils formed within the condensate are distinct from the dilute phase fibril, which suggests that the heterogeneous microenvironment of the LLPS solution can generate structural polymorphs. Understanding the seed-dependent polymorphism in the LLPS-mediated aggregation pathway might open up a huge future research scope in neurodegeneration. Important to note that, the viscoelastic transition of biomolecular condensates can result in spatiotemporal heterogeneity in the condensate microenvironment. Indeed, during the liquid-to-solid transition, condensate from a single protein can progressively convert into multi-phasic architecture with spatial diversity in the molecular property within the condensates.³⁰⁷ The multi-phasic behavior can enormously affect the seeding mechanism of amyloid aggregation as small seeds can be spatially accumulated at different locations within a heterogeneous condensate microenvironment. An interesting observation in this regard is, that the hyperphosphorylated tau generates seeding-competent amyloid aggregates, which can initiate seeding inside and on top of the condensates during liquid-to-solid transition.¹⁶⁰ The condensate interface is particularly efficient in preferentially accumulating fibrillar seeds and enhances amyloid aggregation.^274,308 A curious question would be how the liquid-like condensates maintain spatial control for seed localization and aggregation. A recent theoretical model has explained that even the weak interaction within liquid condensates can effectively sequester fibrillar aggregates in high concentration and the sequestration efficiency increases with the condensate size.³⁰⁹ Moreover, partitioning seeds within condensates may suppress the seed availability in the dilute phase and effectively alter the aggregation kinetics outside the condensate. Future research can delineate the seeding mechanism in the aggregation pathway, which may decipher some intriguing insights into the relevance of secondary nucleation, oligomerization, amyloid polymorphism and toxicity in neurodegeneration.

7. Effect of neurodegeneration-associated factors on aberrant phase separation and amyloid aggregation

Neurons are extremely vulnerable to aberrant phase separation as the postmitotic cells cannot discard pathogenic self-assemblies through cell division and therefore, rely only on the protein-quality control machinery.^310,311 Aging can potentially change the cellular proteostasis machinery with altered protein expression, localization, and degradation processes that hamper the tight regulation required for physiological LLPS.^312–314 Moreover, aging can also affect protein quality control by compromising chaperone activity, receptor binding and ATP production, which in turn affects the condensate regulation.³¹⁵ For example, inhibition of molecular chaperone HSP70 makes stress granule-associated protein (FUS, TDP-43, etc.) susceptible to aberrant liquid-to-solid transition by delaying the post-stress dissolution of stress granules.³¹⁶ ATP is another important regulatory factor in preventing aberrant liquid-to-solid transition, which might act as a biological hydrotrope.³¹⁷ Since neurons are extremely ATP-demanding due to very high metabolic activity, insufficient ATP supply upon aging can potentially hamper protein quality control in neurons, which in turn may promote neuronal protein for aberrant liquid-to-solid transition.³¹⁸ Even though the mechanism for the loss of function and/or gain of toxic function of biomolecular condensates is yet elusive, altered cellular homeostasis in diseased conditions can initiate the formation of pathological phase separation and/or can convert physiological condensate to a pathological one.^245,319,320 For example, the LLPS of FUS is quite critical for initiating DNA damage repair as demonstrated by the failure of the LLPS-deficient FUS variant to catalyze DNA repair at the site of the damage.³²¹ This DNA damage response is impaired in the case of FUS mutation in the nuclear localization signals (NLS), which leads to aberrant liquid-to-solid phase transition and cytoplasmic accumulation of FUS aggregates.^{194,322–324} Similarly, disease-associated hyperphosphorylation of tau inhibits the native function of tubulin polymerization from biomolecular condensate and promotes amyloidogenic solidification of tau condensates.^160,325 Although neurodegenerative diseases are sporadic in many cases, genetic and environmental factors play a huge role in the formation of pathogenic amyloid aggregates.^326,327 Familial mutations,^328–330 gene multiplications,^126,127 deletion,³³¹ and post-translational modifications³³² are key factors associated with amino acid sequence modification, which significantly impact disease onset. Similarly, environmental changes due to metal ions, salt concentration, pH change, small organic molecules, pesticides, RNA, and lipids hugely alter the protein conformation and contribute to aggressive amyloid aggregation.^333–338 Interestingly, the phase separation and aging properties of neurodegenerative disease-associated proteins are also very sensitive to these disease-associated genetic and environmental factors (Fig. 8).^{49,158,339,340} The following section will discuss how common neurodegeneration-associated factors aggravate the pathogenic phase separation leading to amyloid aggregation.

7.1. Genetic factors

All neurodegenerative diseases have shown a direct connection with genetic variations where familial mutations, truncation, and gene multiplication result in early disease progression. For example, early-onset familial mutations have a direct role in AD,^341,342 PD,^343–346 and other neurodegenerative diseases.^347,348 Genetic modification can hugely alter protein surface charge and intermolecular interaction, which impacts liquid-to-solid phase transition probably due to accelerated misfolding of protein, leading to faster amyloid formation. For example, ALS-associated mutations (G156E and R244C) in the FUS proteins result in faster amyloid aggregation kinetics from a condensate, generating fibrillar outbursts from the condensates.²²⁷ Apart from affecting the aging kinetics, the disease-associated mutations can also promote LLPS by bringing down the saturation concentration within the physiological limit, which is otherwise a forbidden event to avoid the risk of protein aggregation. For example, early-onset familial mutants of α-syn such as E46K and A53T, which are known to undergo faster aggregation compared to wild-type, undergo LLPS at lower saturation concentrations and subsequently exhibit faster amyloid aggregation from condensates (Fig. 8c–e).⁴⁷ Similarly, disease-associated point mutants of tau, such as P301L, P301S, ΔK280, and A152T show enhanced phase separation propensity compared to wild type protein, with an accelerated rigidity in droplet dynamics, followed by fibrillar aggregate formation upon prolonged incubation.^160,268 However, another study pointed out that mutations such as P301L, ΔK280, and G272V show similar phase separation propensity as compared to wild-type but only mutants form fibrillar aggregates upon prolonged incubation.³⁴⁹ Thus, disease-associated mutations can specifically promote the amyloid nucleation mechanism without affecting the molecular mechanism associated with LLPS. A similar phenomenon is also observed for the hnRNPA1 protein where pathology-relevant D262V mutant undergoes LLPS with no significant difference as compared to the wild-type but with an enhanced propensity of the mutant to form fibrils.^195,350 On a similar note, familial α-syn mutants A30P, H50Q, A53T, and A53E also exhibit the same LLPS propensity as compared to WT α-syn, except E46K, which significantly promotes LLPS propensity. Interestingly, all these mutants except A53E result in a faster liquid-to-solid transition leading to amyloid aggregation.³⁵¹ Previously, we proposed that enhanced oligomerization propensity and/or their retention might be a shared toxicity mechanism of all α-syn familial mutations, which may contribute to the early onset of PD.³⁴⁵ However, based on these cumulative emerging observations, there is a possibility that faster liquid-to-solid transition might be a prevalently common phenomenon across several disease-relevant proteins, which can be closely associated with oligomerization, toxicity, and disease onset. Further systematic study should be carried out in this direction, which can enormously improve the understanding of pathogenic aggregation. Interestingly, disease-associated mutations of TDP-43, FUS, and hnRNPA1 protein can convert a reversible phase separation into an irreversible one with disease implications.^255,323 Phase separation of these RNA binding proteins is regulated by π–π interactions arising from the regularly interspaced aromatic residues.^169,352,353 This aromatic residue-rich amino acid sequence also forms “low-complexity aromatic-rich kinked segments” (LARKS) that might facilitate phase separation and functional reversible aggregation.³⁵⁴ Interestingly, mutations in the LARK domain reduce the reversible nature of aggregation resulting in irreversible protein aggregates.³⁵⁵ The high fluidity and less viscous nature of TDP-43 ribonucleoprotein (RNP) condensates is indeed essential for faster transport through axon.³⁵⁶ However, ALS-associated mutation rapidly rigidifies these RNP granules affecting the physiological transport in axon and motor neuron activity.^356,357 Moreover, mutant TDP-43 undergoes faster oligomerization and aging kinetics with the emergence of amyloid-like aggregates from the aged condensate.^289,358 Importantly, disease-associated mutations of stress granule-related proteins not only impact phase separation and liquid-to-solid phase transition but also can often impact the relative partitioning within cellular sub-compartments.^359,360 It has been previously reported that ALS/FTD-associated mutation results in the defective nucleocytoplasmic shuttling of protein, which has a direct role in neurodegeneration.³⁵⁹ For example, in the case of the FUS protein, the majority of the ALS/FTD-causing mutations are located either in the PrLD or the protein–tyrosine-rich nuclear signal (PY-NLS) region.^{29,41,361,362} While mutations in the PrLD can accelerate aberrant phase transition and aggregation propensity of FUS, mutations in the PY-NLS region affect the physiological binding of FUS with nuclear transporter receptor, transportin-1 (also known as Karyopherin β2 or Kapβ2).^41,194,363 The inability of Kapβ2 to bind to FUS PY-NLS leads to defective nucleocytoplasmic shuttling resulting in sustained accumulation of aberrant FUS solid-like condensates in the cytoplasm.³⁶⁰ Thus disease-associated mutations not only impede the native location and function of biomolecular condensate but also can initiate aberrant aggregation with implications in neurodegeneration.

Apart from mutation, gene multiplication and overexpression of protein in the compromised proteostasis machinery often elevate the local protein concentration beyond the physiological limit, which is a crucial driving force for pathological phase separation.^195,364 Interestingly, the concentration-dependent change in condensate material property is observed in poly-Q, FUS, and hnRNPA1 where a low concentration regime results in liquid-like dynamic condensates but with very high concentration leads to the formation of a hydrogel composed of amyloid fibrils.^365,366 Misregulated translation and overexpression due to gene multiplication, such as repeat domain gene expansion, which is closely associated with neurodegeneration pathology.³⁶⁷ Interestingly, C9orf72 (chromosome 9 ORF 72) nucleotide repeat expansion, the most common reason for ALS and FTD, results in GR/PR dipeptide repeat expression. The gene multiplication promotes phase separation of hnRNPA1 and TIA1 proteins by decreasing the saturation concentration and also impairs the liquid-like dynamics of the stress granule.³⁶⁸ Similarly, the N-terminal poly-Q repeat expansion of Huntingtin protein can also trigger an aberrant phase transition from liquid-like condensates where fibrous solids are bursting out from aged condensates.²²⁶ Thus, disease-associated genetic variation is emerging as a common factor to promote pathological phase separation and aggregation with its potential role in neurodegeneration.

7.2. Post-translational modification (PTMs)

Post-translational modifications (PTMs) play a crucial role in modulating the protein structure and function in the cellular environment.^369,370 The unregulated PTMs might change the protein's native structure, function, and localization, which often creates protein aggregation and cellular toxicity.^371,372 Moreover, PTMs might alter the conformational stability of proteins, which might impact the LLPS propensity and the material property of the resulting condensates. For example, phosphorylation of tau is important for microtubule stabilization.³⁷³ However, hyperphosphorylation of tau results in not only disruption of the native function but also promotes pathogenic aggregation associated with AD.³⁷⁴ Similarly, 90% of the α-syn population in LB is majorly phosphorylated at Serine residues (S129 and S87), which is a classical hallmark of PD pathology.³⁷⁵ In this direction, it has been shown that phosphorylation of α-syn at S129 is the major form in the composition of LB of the PD brain.^376,377 A phosphomimetic study on S129E demonstrates a decrease in the saturation concentration of α-syn phase separation with faster liquid-to-solid transition into amyloid-like aggregates.⁴⁷ Moreover, intracellular α-syn condensates of the irregular shape, which are formed through LLPS have shown positive staining for S129 phosphorylation in HeLa cells, indicating a strong connection with PD pathology.³⁷⁸ Similar to α-syn, the addition of the negative charges due to hyperphosphorylation decreases the net positive charge of tau and hence enhances its LLPS propensity.^160,268 Moreover, the phosphorylated tau (p-tau 441) droplet exhibits rapid loss in its dynamic behavior and binds to an amyloid cross-β specific dye, thioflavin-S.¹⁶⁰ However, another antibody-specific study showed that in the absence of any cofactors, phosphorylated tau condensates consist of small oligomers, not matured fibrils, and these oligomers are more toxic than the fibrils, which may have direct implications in tauopathies.²⁶⁸ On the other hand, physiological phosphorylation of TDP-43 and FUS has been shown to disrupt the N-terminal oligomerization domain and to reduce LLPS propensity due to the additional negative charge.^169,379 Methylation does not change the overall charge of protein like phosphorylation, rather it alters the charge distribution and steric interaction, which in turn affects the phase separation process.³⁸⁰ Arginine methylation shows a predominant effect on RNA binding proteins such as FUS and hnRNPs as they contain multiple RGG domains of RG-rich motifs.³⁸¹ Arginine methylation suppresses the strength of cation–π interaction, hence performing a physiological role in preventing aberrant phase separation of FUS³⁶³ and hnRNPA2.³⁸² Interestingly, hypomethylated FUS has been shown to form a large number of irregularly shaped condensates with a solid-like material property indicating a potential link with disease pathogenesis.³⁸³ Ubiquitination is another common PTM found in several pathogenic inclusions and is considered a classical biomarker to identify LB in PD pathology.³⁸⁴ Interestingly, ubiquitinated α-syn species have been reported in the condensates of the aged Caenorhabditis elegans population indicating a potential link with the disease mechanism (Fig. 6d).²³³ Ubiquitination has huge physiological importance in protein-quality control machinery by selective proteasomal degradation of misfolded proteins. An interesting study in this regard has shown that site-specific ubiquitination disfavors tau/heparin aberrant condensate formation; while non-specific ubiquitination stabilizes condensates towards aggregation playing a potential pathogenic role.³⁸⁵ N-Terminal acetylation is another physiologically occurring PTM, which prevents the protein from proteasomal degradation and reduces aggregation propensity.³⁸⁶ α-syn shows slow kinetics of phase separation as well as negligible binding with thioflavin-T in N-acetylated form indicating its increased solubility.¹⁵⁸ Similarly, lysine acetylation also disfavors in vitro tau LLPS.³⁸⁷ However, TDP-43 acetylation in the nuclear localizing sequence increases cytoplasmic accumulation of TDP-43 condensates and the mis-localization drives pathogenic cytoplasmic inclusion from condensates.³⁸⁸ These cumulative observations indicate a strong influence of disease-associated PTM in aberrant phase separation and molecular aging.

7.3. Environmental factors

Protein misfolding and aggregation are known to be influenced by various environmental factors.^61,389–392 For example, pH changes, metal ion exposure, and the presence of glycosaminoglycans are known to promote amyloid aggregation of various proteins.^334,390,393 Indeed, various environmental factors are reported as the causative factors associated with major neurological disorders, such as AD and PD.^394–397 Unregulated environmental stress can also impact aberrant LLPS, their material property, and irreversibility. For example, pH change and metal ion binding can switch the native protein conformation to the misfolded states, which not only increases the propensity of LLPS by reducing the saturation concentration but also promotes the amyloid aggregation from condensates. On the other hand, polyanionic species, such as glycosaminoglycans and RNA might interact with positively charged proteins resulting in charge neutralization, which drives rapid phase separation and aggregation. Indeed, protein phase separation and subsequent amyloid aggregation have been shown to be modulated by these environmental factors.^263,320,398 For example, the accumulation of several metal ions, such as Cu²⁺, Fe²⁺, Ca²⁺, Zn²⁺, Al³⁺, Ni²⁺, Co²⁺, and Cr²⁺ have been shown to reduce the saturation concentration of α-syn LLPS and further accelerate the amyloid aggregation kinetics from condensates (Fig. 8c–e).^158,399,400 On the other hand, tau LLPS and solidification are specifically promoted by Zn²⁺ while other transition metal ions, such Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, and Cu²⁺ do not alter the propensity LLPS for tau.^241,401 A recent study has shown that phase separation of Prion protein (PrP) is triggered by Cu²⁺ exposure both in vitro and in live cells across the cell surface, which might act as a protective sequestration mechanism to buffer excess Cu²⁺.⁴⁰² However, oxidative stress induced by prolonged Cu²⁺ or H₂O₂ exposure results in the liquid-to-solid transition in vitro as well as in intracellular condensates into amyloid-rich PrP aggregates. Similarly, PD-relevant exposure to metal toxicants, Cu²⁺ and Fe³⁺ results in the faster transition of liquid-like condensates into amyloidogenic perinuclear solids.⁴⁷ LLPS of α-syn is also enhanced by salts and low pH, which lowers the saturation concentration for phase separation.¹⁵⁸ Charge screening by high salt and low pH between positively charged N-terminal and negatively charged C-terminal exposes the hydrophobic NAC region, which drives the self-assembly prerequisite for α-syn phase separation.^158,403 On the other hand, tau LLPS is primarily driven by electrostatic interactions as the dissolution of the droplets takes place with the addition of 100 mM NaCl.⁴⁰⁴ However, the addition of very high salt concentration shows re-entrant behavior of tau LLPS with quick solidification where the residual hydrophobic interaction drives LLPS with faster droplet aging kinetics.⁴⁰⁵ Apart from pH, salt, and metal ions, glycosaminoglycan (GAGs) can also play a role in amyloid aggregation.^406–410 Heparin, a glycosaminoglycan, is a well-known cofactor that affects tau oligomerization and fibril formation.^409,411,412 This polyanionic cofactor induces molecular self-assembly by reducing the electrostatic repulsion of the positively charged tau. Heparin indeed promotes fibril-like structure formation inside tau phase-separated droplets.^349,413 Moreover, phosphorylated tau in the presence of heparin not only shows accelerated LLPS kinetics but also forms amyloid-like fibrillar aggregates inside droplets.¹⁶⁰ RNA is another important cofactor for regulating LLPS especially for RNA-binding proteins (RBPs) such as FUS, TDP-43, and hnRNPs, where concentration and length of RNA can potentially alter condensate stability, surface tension, and molecular transport properties.^{148,414–418} Interestingly, phase separation of RBP is favored at low RNA concentration while inhibited at high RNA concentration, which is in line with the hypothesis that high RNA abundance in the nucleus inhibits pathological LLPS while aberrant condensation typically occurs in low cytoplasmic RNA concentration.⁴¹⁸ Moreover, condensate viscosity also increases with increasing length of RNA indicating a protein–RNA heterotypic interaction also actively regulates condensate aging.⁴¹⁹ The competing protein–protein and protein–RNA interaction often results in stoichiometry-dependent multiphasic condensate formation recapitulating the complex architecture of in cellulo multicomponent condensates.⁴²⁰ Similar to RNA, ATP is another concentration-dependent regulatory environmental factor for the protein LLPS as its bivalent nature is predominant at low ATP concentrations while hydrophobicity predominates at high concentrations.⁴²¹ At physiological concentration, ATP has been shown to prevent liquid-to-solid phase transition due to site-specific binding with TDP-43 while ALS-associated mutation D169G inhibits its ATP binding and promotes aberrant phase transition.⁴²² Interestingly, α-syn condensate is also transitioned into a percolated mesh-like network structure at 1–5 mM ATP concentration, although the relevance of percolation in disease pathogenesis has to be evaluated in the near future.⁴²³

7.4. Heterotypic protein phase separation and their impact on amyloid aggregation

Coaggregation of multiple neurodegenerative disease-associated proteins into mixed amyloid is a common pathological feature in the various neurodegenerative disease etiology.^424,425 More than 50% of AD patients are reported to exhibit LB; similarly, PD cases are found to develop a high number of tau-containing neurofibrillary tangles (NFT).^426,427 Moreover, α-syn and TDP-43 inclusions were found to co-exist in ALS, FTD, and PD.^428,429 The amyloid deposits of α-syn have also been discovered in patients with CJD, which involves the misfolding of prion protein (PrP).^430,431 Various emerging studies pointed out that α-syn, tau, TDP-43, FUS, and prion proteins can cross-talk and undergo heterotypic phase separation with distinct aging properties as compared to individual phase separation.^{428,432–435} The synergistic complex inter-protein interactions can often show phase separation at lower saturation concentrations than individual protein LLPS and they can lead to faster maturation into ordered solid amyloid species resembling pathological aggregates. For example, electrostatic interactions between the basic N-terminal of PrP and the acidic C-terminal of α-syn promote complex coacervation, which rapidly undergoes aggregation.⁴³³ The bypassing of the lag phase suggests that the synergistic interaction between PrP and α-syn in the complex biomolecular condensates may promote secondary nucleation resulting in a quick liquid-to-solid transition into amyloid-containing condensates. Prp can also form multiphasic condensate with tau driven by electrostatic interaction at a specific protein stoichiometry ratio, which gradually transitioned into amyloid-like co-aggregates upon aging.⁴³² Similar to the tau-PrP system, α-syn can efficiently partition into RNA-induced tau condensate due to the direct binding of the C-terminal of α-syn to the proline-rich region of tau and forms amyloid fibrils upon aging.⁴³⁴ On the other hand, full-length α-syn and tau can also form complex coacervates, without RNA influence, only driven by opposite electrostatic net surface charge.⁴³⁵ Interestingly, the dynamics of small-sized α-syn-tau condensates are quickly arrested by gelation, while the large long-lived multicomponent condensates slowly co-aggregate into amyloid fibrils. This intriguing observation indicates that the intermolecular interaction for amyloid nucleation may not necessarily be the same with condensate formation and/or gelation. α-syn is also reported to modulate the phase behavior of TDP-43-RNA condensates in a multi-component system.⁴³⁶ TDP-43 co-incubated with α-syn showed increased thioflavin-T fluorescence with enhanced aggregation kinetics. Collectively these observations provide a mechanistic underpinning of the synergistic protein–protein interaction during multi-component phase separation, which has a potential connection with neurodegeneration. Although several studies have pointed out the synergistic amyloid aggregation in the multi-component system, few recent reports have shown that the preformed condensates from non-aggregating protein/peptide can accumulate amyloidogenic proteins and exert an inhibitory effect against amyloid aggregation.^309,437,438 For example, the partitioning of the Aβ42 protein into a functional protein condensate has been shown to inhibit the nucleation for amyloid aggregation despite the increase in the local concentration of Aβ42 inside the condensate.⁴³⁷ The observation is very significant and it will open a new possibility of multi-component biomolecular condensate being a protective “safe reservoir” for amyloidogenic proteins. Interestingly, the heterotypic protein–protein interaction in the multicomponent systems can spatiotemporally modulate the aggregation propensity of the partitioning protein. An interesting observation highlights the spatiotemporal modulation of α-syn aggregation within a preformed condensate composed of nonaggregating protein where the condensate interface preferentially promotes its aggregation, but the interior of the condensate exerts a stabilizing effect against aggregation.³⁰⁸ The differential behavior is also dependent on the nature of the partitioning protein where full-length α-syn shows interface-mediated aggregation but the shortest variant comprising aggregation-prone NAC domain specifically accumulates in the condensate interior suppressing its aggregation. The preformed condensate interface can also deplete pathogenic aggregates from the dilute phase, as observed for LLPS of a chaperone protein, p62/SQSTM1, which accumulates Huntingtin diseases-associated polyQ aggregates on its interface for proteasomal degradation.²⁷⁴ Thus, strategically designed multicomponent phase separation can suppress amyloid aggregation for pathogenic proteins, which might open up a huge scope in the therapeutic development of neurodegenerative diseases.

8. LLPS: a generic nucleation mechanism in the amyloid aggregation pathway

Protein misfolding can drive amyloid aggregation with very similar fibrillar properties from the diverse nature of the amino acid sequence, length, and native structures.⁴³⁹ Thus, the amyloid state is considered a highly organized generic state of protein apart from its functional native state.^440,441 Interestingly, LLPS is also emerging as a proteome-wide common phenomenon guided by weak nonspecific multivalent interaction, which is prevalently present in all proteins irrespective of their structural classification.^442–444 In this line, human proteome-wide amino acid sequence analysis revealed that most of the proteins have a propensity for LLPS, which could be accessed, at least transiently, under physiologically relevant cellular conditions.⁴⁴² Moreover, we have recently reported that proteins and polypeptides with various sequences and structural diversity indeed undergo LLPS in the crowded microenvironment further proposing LLPS as a generic phenomenon of protein similar to amyloid state.⁴⁴³ However, there is a fundamental difference between LLPS and amyloid state in their thermodynamic stability as LLPS is highly metastable in nature, inclined towards viscoelastic transition, while the amyloid state represents the most stable state of protein aggregation.⁴⁴⁵ Interestingly, the relative propensity of a protein towards LLPS and amyloid aggregation is enhanced by the same molecular features, namely IDR, LCD, and PrLD. The prevalence of these regions in a protein makes it conformationally flexible facilitating transient multivalent interaction for phase separation and making the protein susceptible to misfolding for amyloid aggregation. Moreover, the molecular crowding within the phase-separated condensates can potentially drive protein misfolding for intrinsically amyloidogenic proteins if they are not under tight proteostasis regulation where chaperone and other cellular factors (e.g., functional PTMs, physiological pH, enzyme and metal binding, etc.) prevent the access to the misfolded pathogenic conformation. Indeed, protein misfolding towards expanded conformation is commonly observed in pathological phase separation of neurodegeneration-related proteins, which initiate the aberrant amyloid aggregation within condensates. Thus, a conceivable hypothesis connecting the dots may propose LLPS as a generic molecular mechanism to promote nucleation for pathogenic amyloid aggregation (Fig. 9).

The classical amyloid fibrils, associated with pathology, are typically irreversible where the stability comes through the steric zipper interaction from hydrophobic residues between the interlocking side chains of the two extended β-sheets.^12,446 Interestingly, amyloid can also be associated with cellular function, represented as “functional amyloid”, which shows reversible dissolution under cellular regulation.^447–450 For example, the low-complexity domain of FUS, TDP-43, and hnRNPA1, which are associated with pathogenic amyloid aggregation, are also capable of forming functional reversible amyloid-like cross-β arrangements, containing LARKS motifs, mainly driven by aromatic residues.³⁵⁴ The striking molecular difference between LARKS and the steric zipper is that the core of the reversible amyloid contains highly dense polar residues that can interact with water; whereas in steric zipper, hydrophobic interlocking amino acids show a dry interface, devoid of water.³⁵⁵ Protein misfolding can unleash the otherwise inaccessible critical hydrophobic interaction dictating the irreversible molecular arrangement with less protein–water interaction. Importantly, LLPS results in the water-poor microenvironment in protein-rich condensate, which is likely to enhance the hydrophobic interactions stabilizing the protein in the misfolded conformation. This is further supported by the observation that prolonged incubation of FUS condensate converts a reversible aggregate into irreversible pathological amyloids.^323,451 Moreover, ALS-associated mutations in the LARKS domain of FUS and TDP-43 can also introduce irreversibility by favoring hydrophobic interaction.³²³ High temperature, high salt, and low pH are often reported to drive faster amyloid formation from condensates, which can be explained by the increase in the residual hydrophobic interaction in these altered conditions.^158,442 Thus, a delicate balance between the solvation energy of hydrophilic and hydrophobic residues of a protein dictates the condensate's reversibility and its aging property towards irreversible amyloid aggregation.⁴⁵² The molecular determinants that drive amyloid aggregation through the condensation pathway require three essential overlapping features: droplet-formation propensity, aggregation-promoting propensity, and multimodal interaction to make an amyloid-like ordered structure.⁵⁹ While weak non-covalent interactions and electrostatic interactions are driving factors for the first two features, hydrophobic interaction is indeed mandatory for molecular ordering towards fibril formation, which is also experimentally supported for low-complexity FUS domain using a range of sophisticated optical techniques.⁴⁵³ Moreover, an intriguing theoretical study further supports the hydrophobicity-driven amyloid aggregation from the condensates for the Aβ42 protein, which has two extra hydrophobic amino acids towards the C-terminus compared to Aβ40 protein and comprises only a small population (∼10%) of the total Aβ content in pathogenic senile plaques.⁴⁵⁴ Based on a sticker-spacer-based model of unfolded Aβ, the authors hypothesized that the small Aβ42 population imparts sufficient non-specific hydrophobicity to drive amyloid-nucleation from Aβ condensates while increasing the population of Aβ40 drives LLPS towards lower saturation concentration. Thus, it is important to note that the molecular interaction prerequisite for LLPS and amyloid aggregation overlap but may not necessarily be the same. While weak multivalent interaction promotes the formation of protein-rich condensates through LLPS, the liquid-to-amyloid transition is mainly driven by enhanced hydrophobicity in the water-poor condensate interior, which might serve as a generic molecular interaction for protein misfolding and amyloid nucleation.

Further, based on the observation that LLPS is commonly observed to initiate amyloid aggregation for several neurodegeneration-associated proteins, an obvious therapeutic strategy therefore could be to target LLPS where inhibition of LLPS and/or inhibition of liquid-to-solid state and fibril formation is possible.⁴⁵⁵ Interestingly, cellular proteostasis machinery utilizes several regulatory mechanisms to prevent amyloid nucleation from condensates, which can inspire the design of the therapeutics.⁴⁵⁶ For example, site-specific functional phosphorylation on an aggregation-prone FUS segment prevents its seeding potential, which can otherwise drive deleterious amyloid aggregation from condensates.³⁰⁵ Functional phosphorylation is also associated with triggering chaperone activity under the surveillance of proteostasis machinery.⁴⁵⁷ Stress-induced phosphorylation of canonical heat shock chaperone proteins Hsp27 leads to preferential enrichment of the chaperone within the FUS condensate inhibiting amyloid aggregation.⁴⁵⁸ Apart from Hsp27, other canonical heat shock chaperons, such as Hsp70 and HSPB1 actively regulate phase separation of stress granule-associated proteins for regulated disassembly with prevention against aberrant aggregation.^242,459 Similarly, pathological phase separation of tau is also inhibited by several molecular chaperones; such as protein disulfide isomerase (PDI), and S100B protein; where disease-associated environmental stress abrogates their recruitment inside condensates.^460,461 Furthermore, O-glycosylation is another emerging regulatory factor for synaptic LLPS and chaperone activity to prevent amyloid aggregation.⁴⁶² These chaperones often bind to oligomers to prevent the nucleation for amyloid aggregation as observed for cellular protein Profilin, which binds to the phase-separated oligomers of Huntingtin N-terminal fragments (Htt-NTFs).⁴⁶³ These cumulative observations strongly suggest that enhancing molecular chaperone expression and/or designing chemical chaperones, chaperone homologs, small molecules, and peptides might inhibit pathological LLPS. Indeed, several chemical chaperones, peptides, and small molecule-based inhibitors that efficiently target the LLPS to prevent amyloid aggregation are reported.^{281,464–466} Recently, a theoretical model has been developed illustrating how to amplify inhibitor efficiency by tuning the condensate volume and partitioning coefficient of the inhibitor, which will increase its effective local concentration within condensates.⁴⁶⁷ Moreover, a remarkable study has revealed that the pharmacodynamic properties of a cancer therapeutic is modulated by the partitioning efficiency of small molecule selectively into the nuclear condensates.⁴⁶⁸ Similarly, small molecules are reported to selectively target a gain-of-function chemo-resistant p53 mutant condensate in the nucleus.^469,470 Thus, designing therapeutics that specifically target nuclear condensates is appearing as a promising therapeutic approach against cancer.⁴⁷¹ An intriguing observation in this regard indicates that the partitioning efficacy of a drug into biomolecular condensates is dictated by its physicochemical property but independent of the molecular target.⁴⁶⁸ The exciting observation implies that drug partitioning efficacy might be considered as an important optimization parameter in future drug design. Indeed, a novel therapeutic strategy, named condensate-modifying therapeutics (c-mods) has been proposed where drug candidates with distinct physicochemical properties target a specific biomolecular condensate and modulate its physical property, stability and/or function as a prevention strategy.⁴⁷² Interestingly, some inhibitors are reported to prevent the aberrant liquid-to-solid transition without affecting the liquid condensate formation, which suggests that pathological fibrillar nucleation from condensates can be decoupled without hampering physiological phase separation by strategic therapeutic design.^464–466 However, the underlying molecular mechanism for stabilizing condensate in a liquid state against amyloid aggregation remains elusive. Extensive future research is essential in this direction, which should include systematic library screening for inhibitor designing and elucidate the inhibitor mechanism at the molecular level.

Apart from neurodegeneration, the emerging amyloid aggregation mechanism preceded by LLPS may also aid in understanding another disease mechanism, such as cancer, which is recently linked with phase separation and amyloid aggregation.^{471,473–477} For example, malignant gain-of-function mutations on tumour suppressor protein p53 have been reported to undergo aberrant phase separation, acting as a precursor for pathogenic amyloid aggregation.⁴⁶⁹ Mis-regulation of physiological LLPS serving important cellular processes such as transcription regulation, signal transduction, oncogenic function, chromatic organization, DNA sensing, and damage response, nuclear organization can effectively contribute to tumorigenesis and cancer.^220,478,479 To take an example, mutations in nucleoporin (NUP98 or NUP214) trigger an oncogenic transcription factor resulting in aberrant biomolecular condensates, which lead to abnormal chromatin structure and tumorigenesis.⁴⁸⁰ Moreover, cancer-associated loss-of-function mutation on another tumour suppressor protein SPOP (speckle-type POZ protein) disrupts the functionally active LLPS and ubiquitination, which might accumulate oncoprotein.⁴⁸¹ Apart from cancer, lethal viral infections such as SARS-CoV-2 (the severe acute respiratory syndrome coronavirus 2) have also shown its potential connection with LLPS and amyloid aggregation as the nucleocapsid protein of the virus contains LCD promoting both events.^482,483 Similarly, a metabolic disorder such as type II diabetes has also been shown to be affected by LLPS-mediated aberrant aggregation as insulin-derived peptides and islet amyloid polypeptide (IAPP) can promote the amyloidogenic pathway from phase separation.^484,485 Since the LLPS phenomenon is ubiquitous in cell physiology, the underlying pathological risk of cellular mis-regulation is an unavoidable consequence leading to diverse disease pathogenesis. Hence elucidating a common pathogenic nucleation mechanism for aberrant protein aggregation might impart significant insight into the disease mechanism. Moreover, the novel amyloid aggregation pathway preceded by LLPS might also help in developing new functional material as the viscoelastic transition is associated with a range of material states. For example, functional amyloid hydrogel formed through LLPS can act as a potential drug delivery system where liquid-like droplets can efficiently entrap drugs while the hydrogel can modulate its release properties. Further, the knowledge from the LLPS-mediated amyloid aggregation pathway can be implemented to rationally design artificial condensates from synthetic polypeptides with interesting molecular features. For example, a very recent study designed an amphiphilic hybrid peptide that combines propensity amyloid formation and phase separation propensity in such a way that the complex condensate shows amyloid fibrillation while maintaining the liquid-like fusion property.⁴⁸⁶ Thus, a range of functional amyloids can be developed from condensates in the near future, which may impart valuable insights into the rational design of supramolecular material with diverse functionality. Moreover, unravelling the molecular mechanism connecting different states of protein self-assembly can open up a huge research scope in synthetic biology; such as enzyme modelling, artificial organelle designing, and biomimetic protocell development that can significantly improve our overall understanding of the origin of life.

Author contributions

Conceptualization – S. K. M.; literature survey – S. M., M. P., K. D. Writing draft; S. M., K. D. Drawings – P. K., M. P., review and editing – S. K. M., S. M, M. P., K. D., P. K. Project administration and supervision – S. K. M.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We acknowledge Shouvik Manna for providing circular dichroism spectroscopy of the α-syn protein. We acknowledge DST SERB-SUPRA (SPR/2021/000103), the Government of India for financial support. S. K. M and K. D. are thankful to Sunita Sanghi Centre for Aging and Neurodegenerative Diseases (SCAN), IIT Bombay, for the financial support.

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