One of the critical definitions of neurodegenerative diseases is the formation of insoluble intracellular inclusion body. These inclusions are found in various neurodegenerative diseases such as Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Huntington’s disease, Parkinson’s disease, and frontotemporal dementia (FTD). Each inclusion body contains disease-specific proteins and is also resistant to common detergent treatments. These aggregates are generally ubiquitinated and thus recognized as misfolded by the organism. They are observed in residual neurons at the affected sites in each disease, suggesting a contribution to disease pathogenesis. The molecular mechanisms for the formation of these inclusion bodies remain unclear. Some proteins, such as superoxide dismutase 1 (SOD1) mutant that causes familial ALS, are highly aggregative due to altered folding caused by point mutations. Still, the aggregates observed in neurodegenerative diseases contain wild-type proteins. In recent years, it has been reported that the proteins responsible for neurodegenerative diseases undergo liquid-liquid phase separation (LLPS). In particular, the ALS/FTD causative proteins such as TAR DNA-binding protein 43 kDa (TDP-43) and fused-in-sarcoma (FUS) undergo LLPS. LLPS increases the local concentration of these proteins, and these proteins eventually change their phase from liquid to solid (liquid-solid phase transition) due to abnormal folding during repetitive separation cycles into two phases and recovery to one phase. In addition to the inclusion body formation, sequestration of essential proteins into the LLPS droplets or changes in the LLPS status can directly impair neural functions and cause diseases. In this review, we will discuss the relationship between the LLPS observed in ALS causative proteins and the pathogenesis of the disease and outline potential therapeutic approaches.
This is a preview of subscription content, access via your institution
Subscribe to Journal
Get full journal access for 1 year
We are sorry, but there is no personal subscription option available for your country.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Al-Chalabi, A. & Brown, R. H., Jr. Finding a treatment for ALS - will gene editing cut it? N Engl J Med 378, 1454-1456 (2018).
Warren, J. D., Rohrer, J. D. & Rossor, M. N. Clinical review. Frontotemporal dementia. BMJ 347, f4827 (2013).
Abramzon, Y. A., Fratta, P., Traynor, B. J. & Chia, R. The overlapping genetics of amyotrophic lateral sclerosis and frontotemporal dementia. Front. Neurosci. 14, 42 (2020).
Renton, A. E. et al. A Hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-Linked ALS-FTD. Neuron 72, 257-268 (2011).
DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-Linked FTD and ALS. Neuron 72, 245-256 (2011).
Vance, C., Rogelj, B., Hortobágyi, T., De Vos, K. J., Nishimura, A. L., Sreedharan, J. et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science https://doi.org/10.1126/science.1165942 (2009).
Sreedharan, J. et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science https://doi.org/10.1126/science.1154584 (2008).
Yan, J. et al. Frameshift and novel mutations in FUS in familial amyotrophic lateral sclerosis and ALS/dementia. Neurology 75, 807-814 (2010).
Hong, M. et al. Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17. Science 282, 1914-1917 (1998).
Goedert, M., Crowther, R. A. & Spillantini, M. G. Tau mutations cause frontotemporal dementias. Neuron 21, 955-958 (1998).
Strong, M. J., Donison, N. S. & Volkening, K. Alterations in Tau metabolism in ALS and ALS-FTSD. Front. Neurol. 11, 598907 (2020).
Li, H. R., Chiang, W. C., Chou, P. C., Wang, W. J. & Huang, J. R. TAR DNA-binding protein 43 (TDP-43) liquid-liquid phase separation is mediated by just a few aromatic residues. J. Biol. Chem. https://doi.org/10.1074/jbc.AC117.001037 (2018).
Boeynaems, S. et al. Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics. Mol. Cell 65, 1044-1055.e1045 (2017).
Kato, M. et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753-767 (2012).
Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell 176, 419-434 (2019).
Patel, A., Lee, H. O., Jawerth, L., Maharana, S., Jahnel, M., Hein, M. Y. et al. A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell 162, 1066-1077 (2015).
Wegmann, S. et al. Tau protein liquid–liquid phase separation can initiate tau aggregation. EMBO J. 37, e98049 (2018).
Iqbal, M. et al. Aqueous two-phase system (ATPS): an overview and advances in its applications. Biol. Proc. Online 18, 18 (2016).
Brangwynne, C. P. et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729-1732 (2009).
Moore, H. M. et al. Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation. Mol. Cell Proteom. 10, M111 009241 (2011).
Gomes, E. & Shorter, J. The molecular language of membraneless organelles. J. Biol. Chem. 294, 7115-7127 (2019).
Ilik, I. A. et al. SON and SRRM2 are essential for nuclear speckle formation. Elife 9, e60579 (2020).
Yamazaki, T. et al. Functional domains of NEAT1 architectural lncRNA induce paraspeckle assembly through phase separation. Mol Cell 70, 1038-1053.e1037 (2018).
Hernandez-Verdun, D. Assembly and disassembly of the nucleolus during the cell cycle. Nucleus 2, 189-194 (2011).
Lamond, A. I. & Spector, D. L. Nuclear speckles: a model for nuclear organelles. Nat. Rev. Mol. Cell Biol. 4, 605-612 (2003).
Protter, D. S. W. & Parker, R. Principles and Properties of Stress Granules. Trends Cell Biol. 26, 668-679 (2016).
Ishihara, T. et al. Decreased number of Gemini of coiled bodies and U12 snRNA level in amyotrophic lateral sclerosis. Hum. Mol. Genet. 22, 4136-4147 (2013).
Aladesuyi Arogundade, O. et al. Nucleolar stress in C9orf72 and sporadic ALS spinal motor neurons precedes TDP-43 mislocalization. Acta Neuropathol. Commun. 9, 26 (2021).
Zhang, P. et al. Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology. Elife 8, e39578 (2019).
Astoricchio, E., Alfano, C., Rajendran, L., Temussi, P. A. & Pastore, A. The wide world of coacervates: from the sea to neurodegeneration. Trends Biochem. Sci. 45, 706-717 (2020).
Pak, C. W. et al. Sequence determinants of intracellular phase separation by complex coacervation of a disordered protein. Mol. Cell 63, 72-85 (2016).
Sing, C. E. & Perry, S. L. Recent progress in the science of complex coacervation. Soft Matter 16, 2885-2914 (2020).
Andre, A. A. M. & Spruijt, E. Liquid-liquid phase separation in crowded environments. Int. J. Mol. Sci. 21, 5908 (2020).
Zhou, H. X. & Pang, X. Electrostatic interactions in protein structure, folding, binding, and condensation. Chem. Rev. 118, 1691-1741 (2018).
Das, S., Lin, Y. H., Vernon, R. M., Forman-Kay, J. D. & Chan, H. S. Comparative roles of charge, pi, and hydrophobic interactions in sequence-dependent phase separation of intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 117, 28795-28805 (2020).
Itoh, Y. et al. 1,6-hexanediol rapidly immobilizes and condenses chromatin in living human cells. Life Sci. Alliance 4, e202001005 (2021).
Lin, Y. et al. Toxic PR poly-dipeptides encoded by the C9orf72 repeat expansion target LC domain polymers. Cell 167, 789-802.e712 (2016).
Martin, E. W. et al. Valence and patterning of aromatic residues determine the phase behavior of prion-like domains. Science 367, 694-699 (2020).
Lee, K.-H. et al. C9orf72 Dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles. Cell 167, 774-788.e717 (2016).
Campen, A. et al. TOP-IDP-scale: a new amino acid scale measuring propensity for intrinsic disorder. Protein Pept. Lett. 15, 956-963 (2008).
King, O. D., Gitler, A. D. & Shorter, J. The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease. Brain Res. 1462, 61-80 (2012).
DePace, A. H., Santoso, A., Hillner, P. & Weissman, J. S. A critical role for amino-terminal glutamine/asparagine repeats in the formation and propagation of a yeast prion. Cell 93, 1241-1252 (1998).
Wang, I. F. et al. The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies. Nat. Commun. 3, 766 (2012).
Alberti, S. et al. A user’s guide for phase separation assays with purified proteins. J. Mol. Biol. 430, 4806-4820 (2018).
Van Lindt, J. et al. A generic approach to study the kinetics of liquid-liquid phase separation under near-native conditions. Commun. Biol. 4, 77 (2021).
Duster, R., Kaltheuner, I. H., Schmitz, M. & Geyer, M. 1,6-Hexanediol, commonly used to dissolve liquid-liquid phase separated condensates, directly impairs kinase and phosphatase activities. J. Biol. Chem. 296, 100260 (2021).
Miyagi, T. et al. An improved macromolecular crowding sensor CRONOS for detection of crowding changes in membrane-less organelles under stressed conditions. Biochem. Biophys. Res. Commun. 583, 29-34 (2021).
Freibaum, B. D., Messing, J., Yang, P., Kim, H. J. & Taylor, J. P. High-fidelity reconstitution of stress granules and nucleoli in mammalian cellular lysate. J. Cell Biol. 220, e202009079 (2021).
Molliex, A. et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123-133 (2015).
Babinchak, W. M. et al. The role of liquid-liquid phase separation in aggregation of the TDP-43 low-complexity domain. J. Biol. Chem. https://doi.org/10.1074/jbc.RA118.007222 (2019).
Kanaan, N. M., Hamel, C., Grabinski, T. & Combs, B. Liquid-liquid phase separation induces pathogenic tau conformations in vitro. Nat. Commun. https://doi.org/10.1038/s41467-020-16580-3 (2020).
Mackenzie, I. R. et al. TIA1 Mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics. Neuron 95, 808-816.e809 (2017).
Schmidt, H. B., Barreau, A. & Rohatgi, R. Phase separation-deficient TDP43 remains functional in splicing. Nat. Commun. 10, 4890 (2019).
Gao, J. et al. Translational regulation in the brain by TDP-43 phase separation. J. Cell Biol. 220, e202101019 (2021).
Levone, B. R. et al. FUS-dependent liquid-liquid phase separation is important for DNA repair initiation. J. Cell Biol. 220, e202008030 (2021).
Reber, S., Jutzi, D., Lindsay, H., Devoy, A., Mechtersheimer, J., Levone, B. R. et al. The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation. Nucleic Acids Res. 49, 7713-7731 (2021).
Maurel, C. et al. Causative genes in amyotrophic lateral sclerosis and protein degradation pathways: a link to neurodegeneration. Mol. Neurobiol. 55, 6480-6499 (2018).
Rosen, D. R. et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59-62 (1993).
Urushitani, M., Kurisu, J., Tsukita, K. & Takahashi, R. Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis. J. Neurochem. 83, 1030-1042 (2002).
Ling, S. C., Polymenidou, M. & Cleveland, D. W. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron 79, 416-438 (2013).
Kraemer, B. C. et al. Loss of murine TDP-43 disrupts motor function and plays an essential role in embryogenesis. Acta Neuropathol. 119, 409-419 (2010).
Arnold, E. S. et al. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.1222809110 (2013).
Dewey, C. M. et al. TDP-43 Is directed to stress granules by Sorbitol, a novel physiological osmotic and oxidative stressor. Mol. Cell. Biol. https://doi.org/10.1128/mcb.01279-10 (2011).
Mitra, J. et al. Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.1818415116 (2019).
Neumann, M. et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130-133 (2006).
Mann, J. R. et al. RNA Binding antagonizes neurotoxic phase transitions of TDP-43. Neuron https://doi.org/10.1016/j.neuron.2019.01.048 (2019).
Asakawa, K., Handa, H. & Kawakami, K. Optogenetic modulation of TDP-43 oligomerization accelerates ALS-related pathologies in the spinal motor neurons. Nat. Commun. https://doi.org/10.1038/s41467-020-14815-x (2020).
Yamanaka, Y., Miyagi, T., Harada, Y., Kuroda, M. & Kanekura, K. Establishment of chemically oligomerizable TAR DNA-binding protein-43 which mimics amyotrophic lateral sclerosis pathology in mammalian cells. Lab. Invest. 101, 1331-1340 (2021).
Nonaka, T. et al. Prion-like properties of pathological TDP-43 aggregates from diseased brains. Cell Rep. 4, 124-134 (2013).
Li, Q., Babinchak, W. M. & Surewicz, W. K. Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43. Nat. Commun. 12, 1620 (2021).
Barmada, S. J. et al. Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis. J. Neurosci. 30, 639-649 (2010).
Charif, S. E., Luchelli, L., Vila, A., Blaustein, M. & Igaz, L. M. Cytoplasmic expression of the ALS/FTD-related protein TDP-43 decreases global translation both in vitro and in vivo. Front. Cell Neurosci. 14, 594561 (2020).
Kuroda, M. et al. Chimeric TLS/FUS-CHOP gene expression and the heterogeneity of its junction in human myxoid and round cell liposarcoma. Am. J. Pathol. 147, 1221-1227 (1995).
Qamar, S. et al. FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-pi Interactions. Cell 173, 720-734.e715 (2018).
Yoshizawa, T. et al. Nuclear import receptor inhibits phase separation of FUS through binding to multiple sites. Cell 173, 693-705.e622 (2018).
Balendra, R. & Isaacs, A. M. C9orf72-mediated ALS and FTD: multiple pathways to disease. Nat. Rev. Neurol. 14, 544-558 (2018).
Lin, Y. et al. Toxic PR Poly-Dipeptides encoded by the C9orf72 repeat expansion target LC domain polymers. Cell 167, 789-802.e712 (2016).
Zhang, Y.-J. et al. Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity. Science 363, eaav2606-eaav2606 (2019).
Maor-Nof, M. et al. p53 is a central regulator driving neurodegeneration caused by C9orf72 poly(PR). Cell 184, 689-708.e620 (2021).
Chen, C. et al. Phase separation and toxicity of C9orf72 poly(PR) depends on alternate distribution of arginine. J. Cell Biol. 220, e202103160 (2021).
Nanaura, H. et al. C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers. Nat. Commun. 12, 5301 (2021).
Shin, Y. et al. Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets. Cell 168, 159-171.e114 (2017).
Gittings, L. M. et al. Symmetric dimethylation of poly-GR correlates with disease duration in C9orf72 FTLD and ALS and reduces poly-GR phase separation and toxicity. Acta Neuropathol. 139, 407-410 (2020).
Solomon, D. A., Smikle, R., Reid, M. J. & Mizielinska, S. Altered phase separation and cellular impact in C9orf72-linked ALS/FTD. Front. Cell Neurosci. 15, 664151 (2021).
Fang, M. Y. et al. Small-molecule modulation of TDP-43 recruitment to stress granules prevents persistent TDP-43 accumulation in ALS/FTD. Neuron 103, 802-819.e811 (2019).
Erdos, G., Pajkos, M. & Dosztanyi, Z. IUPred3: prediction of protein disorder enhanced with unambiguous experimental annotation and visualization of evolutionary conservation. Nucleic Acids Res. 49, W297-W303 (2021).
This work was supported by grants from the JSPS KAKENHI Grant numbers (20H03593 to K. K. and 21H02706 to M. K.). This work was also supported in part by Takeda Science Foundation (K. K.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Kanekura, K., Kuroda, M. How can we interpret the relationship between liquid-liquid phase separation and amyotrophic lateral sclerosis?. Lab Invest 102, 912–918 (2022). https://doi.org/10.1038/s41374-022-00791-x