Manganese promotes α-synuclein amyloid aggregation through the induction of protein phase transition
2021; Elsevier BV; Volume: 298; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2021.101469
ISSN1083-351X
AutoresBingkuan Xu, Shuai Huang, Yinghui Liu, Chun Wan, Yuanyuan Gu, Dianliang Wang, Haijia Yu,
Tópico(s)Parkinson's Disease Mechanisms and Treatments
Resumoα-Synuclein (α-Syn) is the major protein component of Lewy bodies, a key pathological feature of Parkinson's disease (PD). The manganese ion Mn2+ has been identified as an environmental risk factor of PD. However, it remains unclear how Mn2+ regulates α-Syn aggregation. Here, we discovered that Mn2+accelerates α-Syn amyloid aggregation through the regulation of protein phase separation. We found that Mn2+ not only promotes α-Syn liquid-to-solid phase transition but also directly induces soluble α-Syn monomers to form solid-like condensates. Interestingly, the lipid membrane is integrated into condensates during Mn2+-induced α-Syn phase transition; however, the preformed Mn2+/α-syn condensates can only recruit lipids to the surface of condensates. In addition, this phase transition can largely facilitate α-Syn amyloid aggregation. Although the Mn2+-induced condensates do not fuse, our results demonstrated that they could recruit soluble α-Syn monomers into the existing condensates. Furthermore, we observed that a manganese chelator reverses Mn2+-induced α-Syn aggregation during the phase transition stage. However, after maturation, α-Syn aggregation becomes irreversible. These findings demonstrate that Mn2+ facilitates α-Syn phase transition to accelerate the formation of α-Syn aggregates and provide new insights for targeting α-Syn phase separation in PD treatment. α-Synuclein (α-Syn) is the major protein component of Lewy bodies, a key pathological feature of Parkinson's disease (PD). The manganese ion Mn2+ has been identified as an environmental risk factor of PD. However, it remains unclear how Mn2+ regulates α-Syn aggregation. Here, we discovered that Mn2+accelerates α-Syn amyloid aggregation through the regulation of protein phase separation. We found that Mn2+ not only promotes α-Syn liquid-to-solid phase transition but also directly induces soluble α-Syn monomers to form solid-like condensates. Interestingly, the lipid membrane is integrated into condensates during Mn2+-induced α-Syn phase transition; however, the preformed Mn2+/α-syn condensates can only recruit lipids to the surface of condensates. In addition, this phase transition can largely facilitate α-Syn amyloid aggregation. Although the Mn2+-induced condensates do not fuse, our results demonstrated that they could recruit soluble α-Syn monomers into the existing condensates. Furthermore, we observed that a manganese chelator reverses Mn2+-induced α-Syn aggregation during the phase transition stage. However, after maturation, α-Syn aggregation becomes irreversible. These findings demonstrate that Mn2+ facilitates α-Syn phase transition to accelerate the formation of α-Syn aggregates and provide new insights for targeting α-Syn phase separation in PD treatment. α-Synuclein (α-Syn), a cytoplasmic protein abundant in presynaptic nerve terminals, is implicated in Parkinson's disease (PD), dementia with Lewy bodies, and other synucleinopathies (1Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies.Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar, 2Maroteaux L. Campanelli J.T. Scheller R.H. Synuclein: A neuron-specific protein localized to the nucleus and presynaptic nerve terminal.J. Neurosci. 1988; 8: 2804-2815Crossref PubMed Google Scholar, 3Bernal-Conde L.D. Ramos-Acevedo R. Reyes-Hernández M.A. Balbuena-Olvera A.J. Morales-Moreno I.D. Argüero-Sánchez R. Schüle B. Guerra-Crespo M. Alpha-synuclein physiology and pathology: A perspective on cellular structures and organelles.Front. Neurosci. 2019; 13: 1399Crossref PubMed Scopus (41) Google Scholar). While α-Syn monomer is unstructured and has the potential to form amyloid aggregates (4Fauvet B. Mbefo M.K. Fares M.B. Desobry C. Michael S. Ardah M.T. Tsika E. Coune P. Prudent M. Lion N. Eliezer D. Moore D.J. Schneider B. Aebischer P. El-Agnaf O.M. et al.α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer.J. Biol. Chem. 2012; 287: 15345-15364Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 5Theillet F.X. Binolfi A. Bekei B. Martorana A. Rose H.M. Stuiver M. Verzini S. Lorenz D. van Rossum M. Goldfarb D. Selenko P. Structural disorder of monomeric α-synuclein persists in mammalian cells.Nature. 2016; 530: 45-50Crossref PubMed Scopus (525) Google Scholar, 6Nguyen P.H. Derreumaux P. Structures of the intrinsically disordered Aβ, tau and α-synuclein proteins in aqueous solution from computer simulations.Biophys. Chem. 2020; 264: 106421Crossref PubMed Scopus (57) Google Scholar), multiple studies suggested that it forms α-helical tetramers physiologically that resist aggregation (7Bartels T. Choi J.G. Selkoe D.J. α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation.Nature. 2011; 477: 107-110Crossref PubMed Scopus (847) Google Scholar, 8Wang W. Perovic I. Chittuluru J. Kaganovich A. Nguyen L.T. Liao J. Auclair J.R. Johnson D. Landeru A. Simorellis A.K. Ju S. Cookson M.R. Asturias F.J. Agar J.N. Webb B.N. et al.A soluble α-synuclein construct forms a dynamic tetramer.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 17797-17802Crossref PubMed Scopus (346) Google Scholar). α-Syn is involved in neurotransmitter release and many other critical membrane trafficking processes. However, its physiological function has not been completely understood (9Burré J. Sharma M. Tsetsenis T. Buchman V. Etherton M.R. Südhof T.C. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro.Science. 2010; 329: 1663-1667Crossref PubMed Scopus (1119) Google Scholar). In PD, α-Syn forms oligomeric and fibrillar aggregates through the intermolecular assemblies (10Nguyen P.H. Ramamoorthy A. Sahoo B.R. Zheng J. Faller P. Straub J.E. Dominguez L. Shea J.E. Dokholyan N.V. De Simone A. Ma B. Nussinov R. Najafi S. Ngo S.T. Loquet A. et al.Amyloid oligomers: A joint experimental/computational perspective on Alzheimer's disease, Parkinson's disease, type II diabetes, and amyotrophic lateral sclerosis.Chem. Rev. 2021; 121: 2545-2647Crossref PubMed Scopus (141) Google Scholar, 11Agerschou E.D. Schützmann M.P. Reppert N. Wördehoff M.M. Shaykhalishahi H. Buell A.K. Hoyer W. β-Turn exchanges in the α-synuclein segment 44-TKEG-47 reveal high sequence fidelity requirements of amyloid fibril elongation.Biophys. Chem. 2021; 269: 106519Crossref PubMed Scopus (5) Google Scholar). The Lewy bodies and Lewy neurites in the cytoplasm of neurons are mainly composed of aggregated α-Syn (12Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. alpha-Synuclein in Lewy bodies.Nature. 1997; 388: 839-840Crossref PubMed Scopus (5862) Google Scholar, 13Nemani V.M. Lu W. Berge V. Nakamura K. Onoa B. Lee M.K. Chaudhry F.A. Nicoll R.A. Edwards R.H. Increased expression of alpha-synuclein reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis.Neuron. 2010; 65: 66-79Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). While monomeric α-Syn is soluble and not toxic, numerous studies disclose that α-Syn amyloid oligomers and fibrils are the major causes of cellular toxicity (14Karpinar D.P. Balija M.B. Kügler S. Opazo F. Rezaei-Ghaleh N. Wender N. Kim H.Y. Taschenberger G. Falkenburger B.H. Heise H. Kumar A. Riedel D. Fichtner L. Voigt A. Braus G.H. et al.Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase neurotoxicity in Parkinson's disease models.EMBO J. 2009; 28: 3256-3268Crossref PubMed Scopus (341) Google Scholar, 15Volpicelli-Daley L.A. Luk K.C. Patel T.P. Tanik S.A. Riddle D.M. Stieber A. Meaney D.F. Trojanowski J.Q. Lee V.M. Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death.Neuron. 2011; 72: 57-71Abstract Full Text Full Text PDF PubMed Scopus (899) Google Scholar). Many cellular pathway alternations, such as oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, synaptic dysfunction, and autophagy-lysosomal pathway dysfunction, have been linked to the cytotoxic effects of α-Syn aggregates (15Volpicelli-Daley L.A. Luk K.C. Patel T.P. Tanik S.A. Riddle D.M. Stieber A. Meaney D.F. Trojanowski J.Q. Lee V.M. Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death.Neuron. 2011; 72: 57-71Abstract Full Text Full Text PDF PubMed Scopus (899) Google Scholar, 16Fields C.R. Bengoa-Vergniory N. Wade-Martins R. Targeting alpha-synuclein as a therapy for Parkinson's disease.Front. Mol. Neurosci. 2019; 12: 299Crossref PubMed Scopus (96) Google Scholar, 17Wong Y.C. Krainc D. α-Synuclein toxicity in neurodegeneration: Mechanism and therapeutic strategies.Nat. Med. 2017; 23: 1-13Crossref PubMed Scopus (395) Google Scholar, 18Guiney S.J. Adlard P.A. Lei P. Mawal C.H. Bush A.I. Finkelstein D.I. Ayton S. Fibrillar α-synuclein toxicity depends on functional lysosomes.J. Biol. Chem. 2020; 295: 17497-17513Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The aggregations of α-Syn and other amyloid proteins, such as Aβ, tau, IAPP, FUS, and TDP-43, have been studied in vivo and in vitro for decades (10Nguyen P.H. Ramamoorthy A. Sahoo B.R. Zheng J. Faller P. Straub J.E. Dominguez L. Shea J.E. Dokholyan N.V. De Simone A. Ma B. Nussinov R. Najafi S. Ngo S.T. Loquet A. et al.Amyloid oligomers: A joint experimental/computational perspective on Alzheimer's disease, Parkinson's disease, type II diabetes, and amyotrophic lateral sclerosis.Chem. Rev. 2021; 121: 2545-2647Crossref PubMed Scopus (141) Google Scholar, 19Nguyen P.H. Sterpone F. Pouplana R. Derreumaux P. Campanera J.M. Dimerization mechanism of Alzheimer Aβ(40) peptides: The high content of intrapeptide-stabilized conformations in A2V and A2T heterozygous dimers retards amyloid fibril formation.J. Phys. Chem. B. 2016; 120: 12111-12126Crossref PubMed Scopus (34) Google Scholar, 20Ivanova M.I. Lin Y. Lee Y.H. Zheng J. Ramamoorthy A. Biophysical processes underlying cross-seeding in amyloid aggregation and implications in amyloid pathology.Biophys. Chem. 2021; 269: 106507Crossref PubMed Scopus (57) Google Scholar). Protein aggregation and amyloid formation are potential targets for amyloid-related disease (10Nguyen P.H. Ramamoorthy A. Sahoo B.R. Zheng J. Faller P. Straub J.E. Dominguez L. Shea J.E. Dokholyan N.V. De Simone A. Ma B. Nussinov R. Najafi S. Ngo S.T. Loquet A. et al.Amyloid oligomers: A joint experimental/computational perspective on Alzheimer's disease, Parkinson's disease, type II diabetes, and amyotrophic lateral sclerosis.Chem. Rev. 2021; 121: 2545-2647Crossref PubMed Scopus (141) Google Scholar, 16Fields C.R. Bengoa-Vergniory N. Wade-Martins R. Targeting alpha-synuclein as a therapy for Parkinson's disease.Front. Mol. Neurosci. 2019; 12: 299Crossref PubMed Scopus (96) Google Scholar, 17Wong Y.C. Krainc D. α-Synuclein toxicity in neurodegeneration: Mechanism and therapeutic strategies.Nat. Med. 2017; 23: 1-13Crossref PubMed Scopus (395) Google Scholar, 21Doig A.J. Derreumaux P. Inhibition of protein aggregation and amyloid formation by small molecules.Curr. Opin. Struct. Biol. 2015; 30: 50-56Crossref PubMed Scopus (212) Google Scholar, 22Savitt D. Jankovic J. Targeting α-synuclein in Parkinson's disease: Progress towards the development of disease-modifying therapeutics.Drugs. 2019; 79: 797-810Crossref PubMed Scopus (45) Google Scholar). In addition to α-Syn, the Lewy bodies also contain other cellular components, including various organelles and lipids (20Ivanova M.I. Lin Y. Lee Y.H. Zheng J. Ramamoorthy A. Biophysical processes underlying cross-seeding in amyloid aggregation and implications in amyloid pathology.Biophys. Chem. 2021; 269: 106507Crossref PubMed Scopus (57) Google Scholar, 23Shahmoradian S.H. Lewis A.J. Genoud C. Hench J. Moors T.E. Navarro P.P. Castaño-Díez D. Schweighauser G. Graff-Meyer A. Goldie K.N. Sütterlin R. Huisman E. Ingrassia A. Gier Y. Rozemuller A.J.M. et al.Lewy pathology in Parkinson's disease consists of crowded organelles and lipid membranes.Nat. Neurosci. 2019; 22: 1099-1109Crossref PubMed Scopus (331) Google Scholar). Many factors, such as proteins and lipids, coexist with α-Syn and affect its accumulation and cellular toxicity (20Ivanova M.I. Lin Y. Lee Y.H. Zheng J. Ramamoorthy A. Biophysical processes underlying cross-seeding in amyloid aggregation and implications in amyloid pathology.Biophys. Chem. 2021; 269: 106507Crossref PubMed Scopus (57) Google Scholar). For example, the activity of glucocerebrosidase, a lysosomal glycoprotein found in Lewy bodies, affects lipid levels and α-Syn amyloid aggregation (24Muñoz S.S. Petersen D. Marlet F.R. Kücükköse E. Galvagnion C. The interplay between glucocerebrosidase, α-synuclein and lipids in human models of Parkinson's disease.Biophys. Chem. 2021; 273: 106534Crossref PubMed Scopus (16) Google Scholar). Recent studies with Raman spectral imaging revealed that α-Syn fibrils colocalized with lipids in cells (25Watson M.D. Flynn J.D. Lee J.C. Raman spectral imaging of (13)C(2)H(15)N-labeled α-synuclein amyloid fibrils in cells.Biophys. Chem. 2021; 269: 106528Crossref PubMed Scopus (8) Google Scholar), while regulations of α-Syn aggregations by lipids have been previously demonstrated (26Galvagnion C. Buell A.K. Meisl G. Michaels T.C. Vendruscolo M. Knowles T.P. Dobson C.M. Lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation.Nat. Chem. Biol. 2015; 11: 229-234Crossref PubMed Scopus (358) Google Scholar, 27Giehm L. Svergun D.I. Otzen D.E. Vestergaard B. Low-resolution structure of a vesicle disrupting α-synuclein oligomer that accumulates during fibrillation.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 3246-3251Crossref PubMed Scopus (194) Google Scholar, 28Kurochka A.S. Yushchenko D.A. Bouř P. Shvadchak V.V. Influence of lipid membranes on α-synuclein aggregation.ACS Chem. Neurosci. 2021; 12: 825-830Crossref PubMed Scopus (10) Google Scholar). Metal ions such as Cu2+, Zn2+, and Mn2+ are concentrated in the brain and bind to α-Syn (29González N. Arcos-López T. König A. Quintanar L. Menacho Márquez M. Outeiro T.F. Fernández C.O. Effects of alpha-synuclein post-translational modifications on metal binding.J. Neurochem. 2019; 150: 507-521Crossref PubMed Scopus (37) Google Scholar). The C-terminal domain of α-Syn, which contains multiple Asp and Glu residues, can interact with many di- and trivalent metal ions (10Nguyen P.H. Ramamoorthy A. Sahoo B.R. Zheng J. Faller P. Straub J.E. Dominguez L. Shea J.E. Dokholyan N.V. De Simone A. Ma B. Nussinov R. Najafi S. Ngo S.T. Loquet A. et al.Amyloid oligomers: A joint experimental/computational perspective on Alzheimer's disease, Parkinson's disease, type II diabetes, and amyotrophic lateral sclerosis.Chem. Rev. 2021; 121: 2545-2647Crossref PubMed Scopus (141) Google Scholar, 30Binolfi A. Rasia R.M. Bertoncini C.W. Ceolin M. Zweckstetter M. Griesinger C. Jovin T.M. Fernández C.O. Interaction of alpha-synuclein with divalent metal ions reveals key differences: A link between structure, binding specificity and fibrillation enhancement.J. Am. Chem. Soc. 2006; 128: 9893-9901Crossref PubMed Scopus (262) Google Scholar). However, the detailed information for each metal-α-Syn binding is still unclear due to the lack of high-resolution structure data. Manganese is an essential metal involved in many physiological processes (31Pfalzer A.C. Bowman A.B. Relationships between essential manganese biology and manganese toxicity in neurological disease.Curr. Environ. Health Rep. 2017; 4: 223-228Crossref PubMed Scopus (57) Google Scholar, 32Tinkov A.A. Paoliello M.M.B. Mazilina A.N. Skalny A.V. Martins A.C. Voskresenskaya O.N. Aaseth J. Santamaria A. Notova S.V. Tsatsakis A. Lee E. Bowman A.B. Aschner M. Molecular targets of manganese-induced neurotoxicity: A five-year update.Int. J. Mol. Sci. 2021; 22: 4646Crossref PubMed Scopus (13) Google Scholar). However, the rise of manganese utility in the industry has led to increased manganese pollution. The imbalance of manganese homeostasis has the potential to cause neurodegenerative diseases (33Guilarte T.R. Manganese neurotoxicity: New perspectives from behavioral, neuroimaging, and neuropathological studies in humans and non-human primates.Front. Aging Neurosci. 2013; 5: 23Crossref PubMed Scopus (136) Google Scholar, 34Miah M.R. Ijomone O.M. Okoh C.O.A. Ijomone O.K. Akingbade G.T. Ke T. Krum B. da Cunha Martins Jr., A. Akinyemi A. Aranoff N. Antunes Soares F.A. Bowman A.B. Aschner M. The effects of manganese overexposure on brain health.Neurochem. Int. 2020; 135: 104688Crossref PubMed Scopus (35) Google Scholar). Specially, epidemiological investigations indicate that excessive exposure to manganese increases the risk of PD (34Miah M.R. Ijomone O.M. Okoh C.O.A. Ijomone O.K. Akingbade G.T. Ke T. Krum B. da Cunha Martins Jr., A. Akinyemi A. Aranoff N. Antunes Soares F.A. Bowman A.B. Aschner M. The effects of manganese overexposure on brain health.Neurochem. Int. 2020; 135: 104688Crossref PubMed Scopus (35) Google Scholar, 35Gorell J.M. Johnson C.C. Rybicki B.A. Peterson E.L. Kortsha G.X. Brown G.G. Richardson R.J. Occupational exposure to manganese, copper, lead, iron, mercury and zinc and the risk of Parkinson's disease.Neurotoxicology. 1999; 20: 239-247PubMed Google Scholar, 36Lucchini R.G. Martin C.J. Doney B.C. From manganism to manganese-induced parkinsonism: A conceptual model based on the evolution of exposure.Neuromolecular Med. 2009; 11: 311-321Crossref PubMed Scopus (125) Google Scholar). Multiple studies demonstrated that occupational manganese exposure during welding, mining, and other related industrial activities is associated with Parkinsonism (37Racette B.A. McGee-Minnich L. Moerlein S.M. Mink J.W. Videen T.O. Perlmutter J.S. Welding-related parkinsonism: Clinical features, treatment, and pathophysiology.Neurology. 2001; 56: 8-13Crossref PubMed Scopus (306) Google Scholar, 38Jiang Y.M. Mo X.A. Du F.Q. Fu X. Zhu X.Y. Gao H.Y. Xie J.L. Liao F.L. Pira E. Zheng W. Effective treatment of manganese-induced occupational parkinsonism with p-aminosalicylic acid: A case of 17-year follow-up study.J. Occup. Environ. Med. 2006; 48: 644-649Crossref PubMed Scopus (108) Google Scholar, 39Racette B.A. Tabbal S.D. Jennings D. Good L. Perlmutter J.S. Evanoff B. Prevalence of parkinsonism and relationship to exposure in a large sample of Alabama welders.Neurology. 2005; 64: 230-235Crossref PubMed Scopus (150) Google Scholar, 40Rutchik J.S. Zheng W. Jiang Y. Mo X. How does an occupational neurologist assess welders and steelworkers for a manganese-induced movement disorder? An international team's experiences in Guanxi, China part II.J. Occup. Environ. Med. 2012; 54: 1562-1564Crossref PubMed Scopus (7) Google Scholar, 41Rutchik J.S. Zheng W. Jiang Y. Mo X. How does an occupational neurologist assess welders and steelworkers for a manganese-induced movement disorder? An international team's experiences in Guanxi, China, part I.J. Occup. Environ. Med. 2012; 54: 1432-1434Crossref PubMed Scopus (9) Google Scholar, 42Racette B.A. Criswell S.R. Lundin J.I. Hobson A. Seixas N. Kotzbauer P.T. Evanoff B.A. Perlmutter J.S. Zhang J. Sheppard L. Checkoway H. Increased risk of parkinsonism associated with welding exposure.Neurotoxicology. 2012; 33: 1356-1361Crossref PubMed Scopus (105) Google Scholar, 43Mortimer J.A. Borenstein A.R. Nelson L.M. Associations of welding and manganese exposure with Parkinson disease: Review and meta-analysis.Neurology. 2012; 79: 1174-1180Crossref PubMed Scopus (63) Google Scholar, 44Al-Lozi A. Nielsen S.S. Hershey T. Birke A. Checkoway H. Criswell S.R. Racette B.A. Cognitive control dysfunction in workers exposed to manganese-containing welding fume.Am. J. Ind. Med. 2017; 60: 181-188Crossref PubMed Scopus (15) Google Scholar). Long-time environmental exposure to the high level of air manganese may also cause deficits of cognitive function (45Bowler R.M. Kornblith E.S. Gocheva V.V. Colledge M.A. Bollweg G. Kim Y. Beseler C.L. Wright C.W. Adams S.W. Lobdell D.T. Environmental exposure to manganese in air: Associations with cognitive functions.Neurotoxicology. 2015; 49: 139-148Crossref PubMed Scopus (45) Google Scholar, 46Carvalho C.F. Oulhote Y. Martorelli M. Carvalho C.O. Menezes-Filho J.A. Argollo N. Abreu N. Environmental manganese exposure and associations with memory, executive functions, and hyperactivity in Brazilian children.Neurotoxicology. 2018; 69: 253-259Crossref PubMed Scopus (24) Google Scholar). A recent meta-analysis based on previous 22 studies revealed a significantly increased level of circulating manganese in PD patients, further demonstrating that manganese level is a potential risk factor for PD (47Du K. Liu M.Y. Pan Y.Z. Zhong X. Wei M.J. Association of circulating manganese levels with Parkinson's disease: A meta-analysis.Neurosci. Lett. 2018; 665: 92-98Crossref PubMed Scopus (10) Google Scholar). One critical cause of neurotoxicity induced by manganese exposure is the increased α-Syn expression and aggregation (48Sun Y. He Y. Yang L. Liang D. Shi W. Zhu X. Jiang Y. Ou C. Manganese induced nervous injury by α-synuclein accumulation via ATP-sensitive K(+) channels and GABA receptors.Toxicol. Lett. 2020; 332: 164-170Crossref PubMed Scopus (10) Google Scholar, 49Liu Z.Q. Liu K. Liu Z.F. Cong L. Lei M.Y. Ma Z. Li J. Deng Y. Liu W. Xu B. Manganese-induced alpha-synuclein overexpression aggravates mitochondrial damage by repressing PINK1/parkin-mediated mitophagy.Food Chem. Toxicol. 2021; 152: 112213Crossref PubMed Scopus (3) Google Scholar, 50Verina T. Schneider J.S. Guilarte T.R. Manganese exposure induces α-synuclein aggregation in the frontal cortex of non-human primates.Toxicol. Lett. 2013; 217: 177-183Crossref PubMed Scopus (45) Google Scholar, 51Cai T. Yao T. Zheng G. Chen Y. Du K. Cao Y. Shen X. Chen J. Luo W. Manganese induces the overexpression of α-synuclein in PC12 cells via ERK activation.Brain Res. 2010; 1359: 201-207Crossref PubMed Scopus (47) Google Scholar, 52Harischandra D.S. Rokad D. Neal M.L. Ghaisas S. Manne S. Sarkar S. Panicker N. Zenitsky G. Jin H. Lewis M. Huang X. Anantharam V. Kanthasamy A. Kanthasamy A.G. Manganese promotes the aggregation and prion-like cell-to-cell exosomal transmission of α-synuclein.Sci. Signal. 2019; 12eaau4543Crossref Scopus (74) Google Scholar). However, most of these studies were based on cell culture, and their relevance to PD is unknown. Biochemical studies showed that manganese not only shortens the aggregation time of α-Syn but also promotes the formation of large fibrillar aggregates (53Han J.Y. Choi T.S. Kim H.I. Molecular role of Ca(2+) and hard divalent metal cations on accelerated fibrillation and interfibrillar aggregation of α-synuclein.Sci. Rep. 2018; 8: 1895Crossref PubMed Scopus (33) Google Scholar). However, how manganese promotes α-Syn assembly in the early stage remains elusive. Many protein–protein or protein–RNA assemblies can form condensed liquid droplets through weak multivalent interactions, called liquid–liquid phase separation (LLPS). Proteins that can undergo LLPS, such as FUS, TDP-43, and Tau protein, usually contain repetitive domains or intrinsically disordered regions (IDR) (54Li P. Banjade S. Cheng H.C. Kim S. Chen B. Guo L. Llaguno M. Hollingsworth J.V. King D.S. Banani S.F. Russo P.S. Jiang Q.X. Nixon B.T. Rosen M.K. Phase transitions in the assembly of multivalent signalling proteins.Nature. 2012; 483: 336-340Crossref PubMed Scopus (1140) Google Scholar, 55Conicella A.E. Dignon G.L. Zerze G.H. Schmidt H.B. D'Ordine A.M. Kim Y.C. Rohatgi R. Ayala Y.M. Mittal J. Fawzi N.L. TDP-43 α-helical structure tunes liquid-liquid phase separation and function.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 5883-5894Crossref PubMed Scopus (102) Google Scholar, 56Levone B.R. Lenzken S.C. Antonaci M. Maiser A. Rapp A. Conte F. Reber S. Mechtersheimer J. Ronchi A.E. Mühlemann O. Leonhardt H. Cardoso M.C. Ruepp M.D. Barabino S.M.L. FUS-dependent liquid-liquid phase separation is important for DNA repair initiation.J. Cell Biol. 2021; 220e202008030Crossref Google Scholar, 57Ambadipudi S. Biernat J. Riedel D. Mandelkow E. Zweckstetter M. Liquid-liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein tau.Nat. Commun. 2017; 8: 275Crossref PubMed Scopus (308) Google Scholar). Numerous studies identified that a variety of amyloid aggregation-prone proteins initiate aggregations through LLPS (58Wegmann S. Eftekharzadeh B. Tepper K. Zoltowska K.M. Bennett R.E. Dujardin S. Laskowski P.R. MacKenzie D. Kamath T. Commins C. Vanderburg C. Roe A.D. Fan Z. Molliex A.M. Hernandez-Vega A. et al.Tau protein liquid-liquid phase separation can initiate tau aggregation.EMBO J. 2018; 37e98049Crossref PubMed Scopus (368) Google Scholar, 59Tange H. Ishibashi D. Nakagaki T. Taguchi Y. Kamatari Y.O. Ozawa H. Nishida N. Liquid-liquid phase separation of full-length prion protein initiates conformational conversion in vitro.J. Biol. Chem. 2021; 296: 100367Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 60Pytowski L. Lee C.F. Foley A.C. Vaux D.J. Jean L. Liquid-liquid phase separation of type II diabetes-associated IAPP initiates hydrogelation and aggregation.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 12050-12061Crossref PubMed Scopus (22) Google Scholar, 61Xing Y. Nandakumar A. Kakinen A. Sun Y. Davis T.P. Ke P.C. Ding F. Amyloid aggregation under the lens of liquid-liquid phase separation.J. Phys. Chem. Lett. 2021; 12: 368-378Crossref PubMed Scopus (12) Google Scholar). α-Syn has three IDRs and multiple low-complexity domains. Recent studies reported that α-Syn undergoes LLPS to accelerate its intermolecular aggregation, indicating that the phase separation could be an early intermediate stage before fibrillar aggregation (62Ray S. Singh N. Kumar R. Patel K. Pandey S. Datta D. Mahato J. Panigrahi R. Navalkar A. Mehra S. Gadhe L. Chatterjee D. Sawner A.S. Maiti S. Bhatia S. et al.α-Synuclein aggregation nucleates through liquid-liquid phase separation.Nat. Chem. 2020; 12: 705-716Crossref PubMed Scopus (147) Google Scholar, 63Hardenberg M.C. Sinnige T. Casford S. Dada S. Poudel C. Robinson E.A. Fuxreiter M. Kaminksi C. Kaminski-Schierle G.S. Nollen E.A.A. Dobson C.M. Vendruscolo M. Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies.J. Mol. Cell Biol. 2021; 13: 282-294PubMed Google Scholar, 64Hoffmann C. Sansevrino R. Morabito G. Logan C. Vabulas R.M. Ulusoy A. Ganzella M. Milovanovic D. Synapsin condensates recruit alpha-synuclein.J. Mol. Biol. 2021; 433: 166961Crossref PubMed Scopus (20) Google Scholar). In this work, we examined whether Mn2+ regulates α-Syn through LLPS. We observed that α-Syn undergoes LLPS in vitro with the requirement of crowding agents. The addition of Mn2+ efficiently promotes α-Syn liquid-to-solid phase transition, facilitating its amyloid aggregation. Interestingly, Mn2+ could induce α-Syn monomers to form solid-like condensates directly in the absence of crowding agents, bypassing the step of LLPS. We found that Mn2+ could still induce α-Syn phase transition in the presence of lipid membrane. Lipids could be integrated during the formation of condensates but only be recruited to the surface of preformed Mn2+/α-syn condensates. Our studies also demonstrated that Mn2+-induced solid-like condensates proceed with aggregations through recruitment of soluble α-Syn monomers. While it is difficult to disassemble the mature α-Syn fibrillar aggregates, our studies showed that metal chelators could reverse Mn2+-induced aggregates during the phase transition stage. These findings provide a new dimension for understanding the pathogenesis of manganese-induced PD and suggest that LLPS could be a potential target for PD therapy. To study the regulatory effects of Mn2+ on the LLPS of α-Syn, we expressed the full-length human α-Syn (Fig. S1) and examined the conditions in which α-Syn drives LLPS in vitro. We first labeled α-Syn by preparing the EGFP-α-Syn fusion protein. The labeled α-Syn and unlabeled α-Syn were mixed at a molar ratio of 1:9 to form liquid droplets (62Ray S. Singh N. Kumar R. Patel K. Pandey S. Datta D. Mahato J. Panigrahi R. Navalkar A. Mehra S. Gadhe L. Chatterjee D. Sawner A.S. Maiti S. Bhatia S. et al.α-Synuclein aggregation nucleates through liquid-liquid phase separation.Nat. Chem. 2020; 12: 705-716Crossref PubMed Scopus (147) Google Scholar). Using fluorescence microscopy, we discovered that α-Syn underwent LLPS with the requirement of a relatively high protein concentration (Fig. S2) and in the presence of crowding agent PEG-10000 (Fig. S3). Ficoll 400, another macromolecular crowding agent, could also promote α-Syn LLPS, although the efficiency is lower (Fig. S4). By contrast, α-Syn failed to drive LLPS in the presence of small-molecule ethylene glycol, supporting that PEG-10000 acts as a crowding agent to drive α-syn LLPS (Fig. S5). Next, we tested whether Mn2+ plays a role in α-Syn LLPS. In the presence of PEG, a frequently used agent to mimic a crowded cellular environment, 200 μM α-Syn formed the typical smooth liquid droplets. When we included 2 mM Mn2+, the morphology of condensates turned irregular (Fig. 1A). The dose-dependence experiments showed that α-syn could form solid-like condensates when Mn2+ concentration was 400 μM or above (Fig. S6). Then we performed fluorescence recovery after photobleaching (FRAP) experiments to further examine the phase of α-Syn condensates. In the absence of Mn2+, the fluorescence intensity of α-Syn condensates completely returned to their prebleaching state during the 120 s after laser bleaching, indicating the rapid fluidity of α-Syn inside the droplets (Fig. 1, B and C). In contras
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