Phase Separation and Neurodegenerative Disease.pdf

ReviewPhase Separation and NeurodegenerativeDiseases:A Disturbance in the ForceAure lie Zbinden,1,2Manuela Pe rez-Berlanga,1,2Pierre De Rossi,1and Magdalini Polymenidou1,*1Department of Quantitative Biomedicine,University of Z urich,Winterthurerstrasse 190,8057 Zurich,Switzerland2These authors contributed equally*Correspondence:magdalini.polymenidouuzh.chhttps:/doi.org/10.1016/j.devcel.2020.09.014SUMMARYProtein aggregation isthemainhallmarkofneurodegenerativediseases.Many proteinsfoundinpathologicalinclusions are known to undergo liquid-liquid phase separation,a reversible process of molecular self-as-sembly.Emerging evidence supports the hypothesis that aberrant phase separation behavior may serveasatriggerofproteinaggregationinneurodegeneration,andeffortstounderstandandcontroltheunderlyingmechanisms are underway.Here,we review similarities and differences among four main proteins,a-synu-clein,FUS,tau,andTDP-43,whicharefoundaggregatedindifferentdiseasesandwereindependentlyshownto phase separate.We discuss future directions in the field that will help shed light on the molecular mech-anisms of aggregation and neurodegeneration.THE FORCE AWAKENS:AN INTRODUCTION INTO THEFIELD OF LLPSIn a neuronal system far,far away.Protein aggregates are vil-lains of the neurodegeneration Empire,raiding brains and killingneurons.Evading the dreaded aggregation,the phase separa-tion rebellion is a reversible process of molecular self-assemblyfighting for free movement and exchange of molecules,allowingneurons to live long and prosper.However,members of thephase separation rebellion,such as a-synuclein,FUS,tau,andTDP-43 are found in these pathological aggregates.Turmoilhas engulfed the scientific republic when a growing number ofvoices hypothesized that some members of the rebellion maysuccumb to aberrant phase separation behavior and serve thedark aggregation process of the Empire.Thediscoveryofthebroadbiologicalroleofliquid-liquidphaseseparation(LLPS;Table 1)as a true Force connecting most as-pectsofcellularbiology,raisedmanyvoicesandcontroversiesinthe scientific republic over the past decade.LLPS is thereversible unmixing of two macromolecular solutions,creatingtwo separate phases in constant exchange with one another:the dilute phase and,contained within it,the condensed phase(Alberti et al.,2019;Boeynaems et al.,2018).Phase transitionis driven by the minimization of the global free energy of themacromolecular solution,which is achieved by maximizingweak inter-and intramolecular interactions between the consti-tuting macromolecules.This phenomenon only occurs at acertain solubilization limit,known as the saturation concentra-tion,which is characteristic for each phase-separating system(Alberti et al.,2019).Whether a system undergoes LLPS de-pends not only on the molecular identity and solution concentra-tionofthebiopolymer(Table1)butalsoondiverseenvironmentalvariables,such as temperature(Cinar et al.,2019),salt type andconcentration(Wolf et al.,2014),co-solutes(Brangwynne et al.,2009;Hayesetal.,2018),andpH(Adame-Aranaetal.,2019;Ruffet al.,2018).However,the ability to undergo LLPS has been sug-gested to be a common property of all biopolymers under spe-cific conditions,regardless of their sequence(Alberti et al.,2019;Banani et al.,2017).Inrecentyears,LLPShasbeenproposedtoexplaintheforma-tion and dynamic behavior of a variety of cellular membranelesscompartments,formed by proteins binding to nucleic acids orother polypeptides acting like polymeric scaffolds(Table 1)(Ba-nani et al.,2016).Some of these structures,like the multiple sub-compartments within the nucleus,are common to all cells.Thenuclear speckles,paraspeckles,nucleoli,or Cajal bodies are ex-amples of such nuclear structures,some of which weredescribed as early as the 19th century(Cajal,1903;Wagner,1835).However,LLPS-driven membraneless compartmentsare not exclusively nuclear.The processing bodies(P-bodies)(Sheth and Parker,2003)or the stress granules(SGs)(Kedershaet al.,1999),composed by pools of translation-stalled mRNAs,are formed in the cytoplasm.Highly specialized condensatesare actually specific to some cell types.For example,neuronspresent membraneless compartments that are unique to thesynapses(Wu et al.,2019a;Zeng et al.,2018)and axonal com-partments(Liao et al.,2019).Most of these functional substructures share certain featuressuchasamembranelesssphericalshapedefinedbysurfaceten-sion,a dynamic behavior,and an assembly mechanism(Albertiet al.,2019;Brangwynne et al.,2009,2011),while they differ incomposition,subcellular localization,and function(Decker andParker,2012).They are usually referred to as droplets,speckles,granules,bodies,compartments,foci,densities,puncta,orclus-ters.However,due to their ability to highly concentrate andcondense specific assemblies of biomolecules in discretecellular sites,an accepted overarching term is biomolecularcondensate(Table1)(Bananietal.,2017;Choietal.,2020).Bio-molecular condensates can influence a myriad of biochemicalconcepts including reaction kinetics(Kuznetsova et al.,2015;llDevelopmental Cell 55,October 12,2020 2020 Published by Elsevier Inc.45Table 1.GlossaryBiomolecularcondensateMembraneless cellular compartment concentrating a certain set of multivalent biopolymers,formed through LLPS(Banani et al.,2017;Brangwynne et al.,2009,2011).Despite presenting different localization,composition,andfunctionality,they share similar morphology,dynamics,and assembly processes(Banani et al.,2017).BiopolymerNatural macromolecules composed of repeated monomeric units linked by covalent bonds produced by livingorganisms from cellular matter.There are three classes of biopolymers as defined by their monomeric unit andstructure:polypeptides,polynucleotides,and polysaccharides(Choi et al.,2020).ComplexcoacervationProcess involving interactions among oppositely charged polyions,which leads to phase separation(Aumiller andKeating,2016;Overbeek and Voorn,1957;Pak et al.,2016).In biological systems,complex coacervation is knownto occur between proteins with highly opposed net charges(Bhopatkar et al.,2020;Pak et al.,2016)or positivelycharged proteins and nucleic acids(Aumiller and Keating,2016;Banerjee et al.,2017;Lin et al.,2019;Zhang et al.,2017).Phase separation of the oppositely charged polyions is mainly driven by electrostatic interactions that lower the netcharge and result in a polymer-rich dense phase(Veis,2011).Nevertheless,hydrophobic and dipole interactions,as well as nucleic acid conformation,also play a role in this mechanism(Timilsena et al.,2019),by generatingadditional interactions.IDRPolypeptide segment that is unlikely to adopt a defined,fixed 3D structural conformation,as opposed to structureddomains.As such,they are characterized by protein-folding energy landscapes lacking the well-defined,funnel-shaped energy minimum of folded domains.They instead present a hilly plateau,resulting from their varied setof possible conformations.IDRs generally lack bulky hydrophobic amino acids that allow building well-organizedhydrophobic cores,and their functions,if described,thus arise through different interactions and dynamics(Uversky,2013).IDRs are involved in various interactions with partners of all types in many biological processes like molecularrecognition,cell-cycle control,and signaling(Piovesan et al.,2017;Vucetic et al.,2007;Wright and Dyson,2015).They are highly abundant in the proteome and found in all domains of life(Uversky,2010;Xue et al.,2012).LLPSReversible process by which a solution of biopolymers(proteins or nucleic acids)separates into two coexistingphases:a condensate phase with liquid-like properties,and a dilute phase.LLPS is mediated by weak intermolecularinteractions in conditions where biopolymer-biopolymer and solvent-solvent interactions are energetically favoredover biopolymer-solvent interactions.Noncovalent interactions act like physical crosslinks building a biopolymernetwork with varied material properties ranging from liquid to solid behavior(Alberti et al.,2019;Boeynaems et al.,2018;Choi et al.,2020).LCRPolypeptide segments with a biased amino acid frequency distribution as compared with the amino acidproportions found in the proteome,and mostly predicted to structurally be IDRs(Mier et al.,2020).PrLDSubcategory of LCRs enriched in glycines and uncharged polar amino acids(Ser,Tyr,Gln,and Asn)and showingsequence similarities to yeast prion proteins(Alberti et al.,2009).PrLDs are often found in aggregation-prone RBPsassociated with neurodegenerative disorders,such as TDP-43 and FUS(Alberti et al.,2009;King et al.,2012).NeurodegenerativediseasesIncurable,age-dependent diseases presenting irreversible and progressive loss of selective neuronal populations.Neurodegenerative diseases are diverse in pathobiology,causing a diversity of impairments including memory loss,cognitive changes,and loss of mobility,speech,or breathing(Gitler et al.,2017).Neurodegenerative diseases areclassified based on clinical presentation,anatomic distribution,or molecular abnormalities.Despite their diversity,they are believed to share key pathological processes leading to neuronal dysfunction and death(Dugger andDickson,2017).Nuclear importreceptorsSpecialized proteins that bind the NLS of cargo proteins in the cytoplasm in order to transport them into thenucleus through the nuclear pore complex(NPC).Nuclear porecomplexGroup of proteins spanning the double nuclear membrane thereby forming channels(pores)that enable communicationbetween the nucleoplasm and the cytoplasm,mostly focused in the transport of biopolymers like proteins and RNA.In recent years,impaired nucleocytoplasmic active transport and passive diffusion due to alterations in the NPC andthe nuclear membrane integrity has emerged as a key feature of a wide range of neurodegenerative diseases,suchas ALS,FTD,or Huntingtons disease(Kim and Taylor,2017;Li and Lagier-Tourenne,2018).OligomerizationSpecific,reversible,noncovalent intermolecular interactions of monomeric subunits resulting in a macromolecularcomplex.As opposed to polymers(such as actin and tubulin),oligomers have a defined number of monomers perassembly.Oligomers differ from biomolecular condensates in size,stoichiometric restriction,and temporal dynamics(Banani et al.,2017).In contrast to pathological oligomerization,which refers to the association of misfolded speciesforming precursors of large pathological assemblies,physiological oligomerization involves well-folded native domains(Mizuno and Kawahara,2013)that are often important for protein functionality.Pathological oligomers can also differfrom each other in size,b sheet content and exposed hydrophobicity(Bengoa-Vergniory et al.,2017).The distinct rolesof functional and pathological oligomers in disease are often muddled and better distinction of the species is requiredgoing forward.This necessitates better definition of the structural and functional features of the oligomers,which isoften missing.Pathologicalassembliesor aggregatesThe terms aggregates and pathological assemblies are often used interchangeably in the literature.Pathologicalproteinaceous assemblies are hallmarks of protein conformation diseases(or proteinopathies),which includeneurodegenerative diseases(Westermark et al.,2002).Pathological assemblies are formed by a still unclear molecular(Continued on next page)ll46Developmental Cell 55,October 12,2020ReviewLi et al.,2012),specificity of biological processes(Decker andParker,2012;OConnell et al.,2012;Prouteau and Loewith,2018),buffering(Eldar and Elowitz,2010),adaptive filtration(Schmidt and Go rlich,2016),and mechanical force generation(Bergeron-Sandoval et al.,2018).These physicochemical prop-erties suggest a wide range of possible cellular functionalities forbiomolecular condensates because they enable a tight spatio-temporal control of biochemical reactions,such as the essentialcellular functions of cell differentiation(Liu et al.,2020;Quirozet al.,2020),cytoskeletal regulation(Case et al.,2019;Herna n-dez-Vegaet al.,2017),and metabolic control(Prouteau and Loe-with,2018).Becausetheylackmembranes,thesepotentialfunc-tions are switched on and off by the formation and disassemblyof the condensates in reaction to small environmental changeslike temperature or pH changes(Lin et al.,2015;Molliex et al.,2015;Nott et al.,2015).Their membraneless nature additionallyallows for a rapid movement of molecules between phases,without the delay from transportation over a barrier,enablingfastchemicalequilibriumbetweensubcompartmentsofdifferentproperties and their assembly and disassembly occurs indepen-dently from any energy input(Griffin et al.,2011).Due to thisquick responsiveness,biomolecular condensates are regardedas a faster version of cellular regulation when compared withtranscriptional and translational control(Banani et al.,2017;Rib-ack et al.,2017).Ultimately,LLPS is widely considered a key physiological pro-cess that allows for the dynamic biogenesis of at least a subsetof essential membraneless organelles.While repeat RNA pre-sents phase separation properties(Jain and Vale,2017)thatmay be relevant in neurodegenerative disorders,this reviewwill focus on polypeptide LLPS.In recent years,key discoveriesonbiomolecularcondensateshavelinkedtheprocessofLLPStoprotein aggregation in neurodegenerative diseases,improvingboth our biophysical knowledge of LLPS and our understandingof the pathology.Indeed,an increasing set of proteins that canphysiologically undergo LLPS are found in pathological aggre-gates(Table 1).Protein aggregates are pathognomonic forseveral human disorders,most notably neurodegenerative dis-eases(Table 1),including amyotrophic lateral sclerosis(ALS),frontotemporal dementia(FTD),Alzheimers disease(AD),andParkinsons disease(PD).This has led to the hypothesis thatLLPS may favor the formation of aggregates(reviewed in Liet al.,2013;Polymenidou and Cleveland,2011),and a flurry ofstudies has focused on understanding the behavior of neurode-generative-disease-associatedproteinswithinbiomolecularcondensates both in vitro and in cells.In this review,we discussthe current understanding of LLPS behavior of four such pro-teins:a-synuclein,FUS,TDP-43,and tau(Figure 1).Despiteapparent structural and functional differences(Figure 2),wedescribe shared properties that could underlie their ability toTable 1.Continuedmechanism that involves the self-assembly of misfolded protein species,which assemble into insoluble proteinaggregates that may be amorphous,filamentous,or amyloid like(Dobson,2017).The different disease-associatedassemblies contain,predominantly,but not exclusively,a specific protein,which is unique to the disorder or asubtype thereof(Grundke-Iqbal et al.,1986a;Neumann et al.,2006,2011;Spillantini et al.,1997),and may present anabnormal pattern of posttranslational modifications(Tables S2S5)and other alterations,such as disulfide bridges(Cohen et al.,2012;Furukawa et al.,2006,2011).Several neurodegenerative-disease-associa