The Mechanism of Protein Condensation in Neurodegenerative Diseases

Muya Zhang; Jiaqi Liu; Wang Chen; Liqiang Wang; Jie Chen; Yi Liang

doi:10.7536/PC220534

Progress in Chemistry ›› 2022, Vol. 34 ›› Issue (7) : 1619-1625 DOI: 10.7536/PC220534

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The Mechanism of Protein Condensation in Neurodegenerative Diseases

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Abstract

Protein and RNA molecules tend to form supramolecular assemblies called membrane-less organelles via liquid-liquid phase separation of proteins in cells. These organelles have fusion properties similar to liquid droplets formed by biological macromolecules when their concentrations are higher than saturation concentrations. Upon aging, these dynamic droplets change their material properties and transform into gels, followed by formation of solid condensates. It is well known that proteins with low-complexity domains undergo liquid-liquid phase separation. The common pathological feature of neurodegenerative diseases such as transmissible spongiform encephalopathy, amyotrophic lateral sclerosis, and Alzheimer’s disease is toxic oligomers or amyloid aggregates formed by misfolded proteins including prion protein, DNA- and RNA-binding protein TDP-43, and Tau protein. A large number of studies have shown that prion protein, TDP-43, and Tau protein all undergo liquid-liquid phase separation and form protein condensates. This review summarizes the role of protein phase separation and condensation in neurodegenerative diseases, elaborates the mechanism of protein condensation modulating transmissible spongiform encephalopathies, TDP-43 proteinopathies, and tauopathies, and focuses on the initiation effect of phase separation on aggregation and toxicity of misfolded proteins in neurodegenerative diseases. Finally, we discuss and prospect the challenges and opportunities of association study between protein condensation and neurodegenerative diseases.

Contents

1 Introduction

2 liquid-liquid phase separation and condensation of prion proteins

3 liquid-liquid phase separation and condensation of TDP-43

4 liquid-liquid phase separation and condensation of Tau protein

5 Discussion

6 Challenge and outlook

Graphical abstract

Keywords

protein phase separation / neurodegenerative diseases / prion protein / TDP-43 / Tau / condensate

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Muya Zhang, Jiaqi Liu, Wang Chen, Liqiang Wang, Jie Chen, Yi Liang. The Mechanism of Protein Condensation in Neurodegenerative Diseases. Progress in Chemistry, 2022, 34(7): 1619-1625 DOI:10.7536/PC220534

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1 Introduction

Highly compartmentalized eukaryotic cells have evolved a series of organelles, such as endoplasmic reticulum, mitochondria, lysosomes and Golgi apparatus, to ensure that each component of the cell accurately and subtly performs specific biological functions in both spatial and temporal dimensions and maintains cell homeostasis^[1]. In addition, biomacromolecules such as proteins and RNA in the cytoplasm can be assembled into membrane-less supramolecular assemblies through liquid-liquid phase separation, which are called membrane-less organelles, including Cajal bodies in the nucleus, P-bodies in the cytoplasm and stress granules. These membraneless organelles have the same compartmentalized components and morphology as membranous organelles, and participate in a variety of physiological functions such as cell signal transduction, gene expression regulation and environmental stress^{[2⇓⇓⇓~6]}. Membraneless organelles have fusion properties similar to droplets. When the concentration exceeds the saturation concentration, these biological macromolecules will form droplets, then transform to the gel state, and finally form solid aggregates^[6⇓⇓~9]. Proteins that can undergo phase separation are known to have similar structural features, namely low complexity domains rich in charged amino acids and polar amino acids or glycine^{[6,10⇓ ~12]}. When solution conditions favor the formation of large-scale dynamic intermolecular interactions, proteins with low-complexity domains will stratify into light and dense phases, a process known as liquid-liquid phase separation^[13].

A common pathological feature of neurodegenerative diseases such as transmissible spongiform encephalopathy (transmissible spongiform encephalopathy), Amyotrophic lateral sclerosis (Amyotrophic lateral sclerosis), and Alzheimer's disease (Alzheimer's disease) is the formation of toxic oligomers or amyloid fibrils by misfolded proteins including prion protein, TAR DNA-binding protein-43 (DNA- and RNA-binding protein TDP -43), and Tau protein^{[11,12,14⇓ ~16]}. A large number of studies have shown that prion proteins, TDP-43 and Tau proteins can undergo liquid-liquid phase separation. On the one hand, the formation of these droplets is likely to be an initial step in the misfolding and accumulation of these proteins. On the other hand, some pathogenic proteins themselves are components of membraneless organelles, such as TDP-43, which functions in RNA transcription and alternative splicing^{[17⇓⇓⇓⇓⇓⇓⇓~25]}^{[26⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓~41]}^{[11,41⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ ~57]}^{[6,10⇓ ~12,42]}^[58,59]. Errors in the assembly, disassembly, or clearance of these functional components can lead to the formation of pathological aggregates. There is no doubt that the study of the relationship between important proteins related to neurodegenerative diseases and phase separation has broad scientific prospects. In this review, we focused on the mechanisms of protein phase separation in the regulation of neurodegenerative diseases such as transmissible spongiform encephalopathies, TDP-43 proteinopathies and Tau proteinopathies, and discussed the induction of prion protein, TDP-43 and Tau protein to form oligomers and amyloid fibrils by phase separation.

2 Liquid-liquid phase separation and coacervation of prion protein

Transmissible spongiform encephalopathies, also known as prion diseases, are transmissible, fatal neurodegenerative diseases caused by prion protein (PrP) misfolding^{[14,60⇓⇓ ~63]}. Prusiner S B, winner of the 1997 Nobel Prize in Physiology or Medicine, first described prions, protein infectious agents^[60,61]. The main pathological feature of transmissible spongiform encephalopathies is that prion protein changes from cellular PrP^C to pathological PrP^Sc,PrP^Sc, which can act as a template to interact with PrP^C and induce its conformational transition^{[60⇓⇓~63]}^[60,62,63]. The structures of PrP^Sc and PrP amyloid fibrils have been resolved, and scientists have gradually revealed the conformational conversion mechanism of prion pathogenesis, and found that the C-terminal of prion protein changes from a spherical structure rich in α-helix to a lamellar structure rich in β-sheet during the pathogenesis^[64⇓~66]. Full-length wild-type human PrP and its pathological mutant E196K fibrils are both composed of two strands of fibrils wound in a left-handed helix. The former interacts through two pairs of salt bridges to form a short hydrophilic fibril interface, and the fiber core is composed of 170 to 229 at its C terminus and contains six β-sheet structures (from β1 to β6) (Fig. 1A)^[65]. The latter interacts through four pairs of salt bridges to form a long hydrophilic and hydrophobic fibril interface, and the core of the fibril is composed of 175 to 217 at its C terminus, containing five β-sheet structures (from β1 to β5) (Figure 1B)^[66]. The 263K hamster prion fiber is composed of a single strand of fibrils wound in a left-handed helix, with six β-sheet structures (from β1 to β6) at the N-end (green) and five β-sheet structures (from β7 to β11) at the C-end (magenta) (Fig. 1C)^[64].

Researchers have found that PrP can undergo liquid-liquid phase separation to form aggregates, which gradually confirms that liquid-liquid phase separation is closely related to PrP aggregation^{[17⇓⇓⇓⇓⇓⇓⇓~25]}. Kostylev et al. First observed the phenomenon of liquid-liquid phase separation and condensation of PrP, and used nuclear magnetic resonance spectroscopy to prove that the C-terminal α2 and α3 of PrP would unfold and form a disordered structure during the phase separation process^[18]. Then, our laboratory found that the PrP protective mutation G127V significantly inhibited the liquid-liquid phase separation and aggregation of PrP, and further introduced an amino acid (Ile) with a different hydrophobic chain at the same position, and found that G127I PrP had a similar protective effect as G127V PrP^[19]. The results showed that the two neutralizing mutants (G127V PrP and G127I PrP) significantly reduced the cytotoxicity caused by PrP fibril formation, mitochondrial damage and ROS elevation^[19]. At the same time, Matos et al. Found that DNA promoted PrP liquid-liquid phase separation in a concentration-dependent manner and promoted PrP fibrillization and aggregation^[20]. A year later, Tange et al. Observed that PrP could undergo a liquid-solid phase transition during liquid-liquid phase separation, in which PrP produced amyloid aggregates rich in β-sheets and resistant to proteases^[21]. Then, Agarwal et al. Found that PrP pathological mutant Y145 Stop was more prone to liquid-solid phase transition than wild type PrP, and PrP amyloid fibers were detected in the solid phase by atomic force microscopy^[22]. Recently, the laboratory observed that the N-terminal disordered structure of PrP and the C-terminal acidic region of α-synuclein form droplets through electrostatic interaction, the intervention of RNA regulates the transition of phase separation from liquid to solid, and the change of droplet state drives the protein complex to form amyloid fibrils^[25].

To sum up, the C-terminal domain, N-terminal disordered structure and RNA of PrP play an important role in regulating its liquid-liquid phase separation and aggregation, so the regulation of PrP liquid-liquid phase separation and aggregation is a potential research direction.

3 Liquid-liquid phase separation and coacervation of TDP-43

Amyotrophic lateral sclerosis, also known as amyotrophic lateral sclerosis, is a progressive neurodegenerative disease involving major motor neurons in the cerebral cortex, brain stem, and spinal cord.One of the pathogenic mechanisms is that TAR DNA-binding protein-43 (TDP-43) and copper-zinc superoxide dismutase (SOD1) form fibrillar aggregates in motor neurons of the central nervous system, as well as in astrocytes^[15,67]^{[15,67⇓ ~69]}. TDP-43 belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which is a DNA/RNA-binding protein mainly expressed in the nucleus. It can shuttle to the cytoplasm and interact with other related proteins to form various ribonucleoprotein complexes such as stress granules^[70]. TDP-43 has become one of the research hotspots of neurodegenerative diseases since Neumann et al found that TDP-43 accumulated abnormally in the cytoplasm of damaged neurons and glial cells in patients with amyotrophic lateral sclerosis and frontotemporal dementia in 2006^[71].

The researchers found that TDP-43 can undergo liquid-liquid phase separation to form aggregates, and this process is closely related to the pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia^{[26⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓~41]}. Studies have shown that there is a glycine-rich low-complexity domain in the C-terminal domain of TDP-43, and almost all mutations associated with amyotrophic lateral sclerosis and frontotemporal dementia occur in this region, which is also a region of current research focus^{[26⇓⇓~29]}. Through extensive mutagenesis of TDP-43, Li et al found that three tryptophans (Trp334, Trp-385, and Trp-412) located in the C-terminal domain were very important for the liquid-liquid phase separation of TDP-43, among which Trp334 in the C-terminal α-helix region was the most important, and proposed that the formation of condensates by TDP-43 might only be regulated by a small number of key amino acid residues^[26]. Hallegger et al. Recently found that the conserved sequence located at the 316-326 position of the C-terminal domain had the most obvious effect on the formation of aggregates by liquid-liquid phase separation of TDP-43^[27]. The phase separation ability of TDP-43 truncations with deletion of this sequence was significantly reduced in vitro and in cells, and the point mutation in this region also affected the phase separation ability of the C-terminal domain, suggesting that this sequence plays an important role in the regulation of TDP-43 condensation^[27]. The α-helical region located in the C-terminal domain is a short and unique tunable module that is important for TDP-43 to perform both liquid-liquid phase separation and RNA splicing functions^[28]. Pantoja-Uceda et al. Found that the Phe-Gly motif is a key element of the TDP-43 C-terminal domain, which is important for TDP-43 liquid-liquid phase separation to form aggregates and further grow fibers^[29].

In addition, other domains of TDP-43 are also closely associated with its liquid-liquid phase separation. A large number of studies have shown that the N-terminal domain is indispensable for TDP-43 to perform functions such as RNA transcription and alternative splicing, and is also involved in the pathological accumulation of TDP-43^[15,67]. Wang et al. Found that the mimic phosphorylation of Ser at position 48 in the N-terminal domain significantly inhibited the liquid-liquid phase separation of TDP-43, and also reduced the RNA splicing activity of TDP-43^[30]. In addition to the C-terminal domain and the N-terminal domain, TDP-43 also contains two RNA recognition domains, RRM1 and RRM2, in which RRM1 plays a leading role in the binding of TDP-43 to UG-rich RNA or TG-rich DNA, while RRM2 plays a supporting role^[72]. Wang et al. Found that TDP-43 forms membraneless, droplet-like, highly dynamic and reversible TDP-43 nuclear bodies with cytoprotective function in the nucleus through liquid-liquid phase separation during cell stress. RRM1 and RRM2 play different and even antagonistic "centripetal-centrifugal" roles, so TDP-43 nuclear bodies lacking RRM1 or RRM2 show interesting "nuclear" or "ring" morphology, respectively^[31].

Post-translational modification is a key regulator of TDP-43 liquid-liquid phase separation. In 2021, Yu et al. Reported in Science that RNA-binding defective TDP-43 can drive the protein to decompose into a liquid spherical shell and a liquid core^[33]. Acetylation modification, molecular chaperone Hsp70, and proteasome activity coordinately regulate the liquid-liquid phase separation of TDP-43 and the conversion to gel and solid condensates^[33]. In contrast, casein kinase 1δ-mediated TDP-43 hyperphosphorylation significantly inhibited the liquid-liquid phase separation and accumulation of TDP-43 in membraneless organelles, and thus TDP-43 hyperphosphorylation may be a protective cellular response that inhibits TDP-43 aggregation^[36]. In addition, specific RNA can also affect the liquid-liquid phase separation process of TDP-43, for example, the long non-coding RNA NEAT1 in neurons significantly promotes the occurrence of liquid-liquid phase separation of TDP-43 and the formation of TGP-43 nuclear bodies in stressed cells, which indicates that NEAT1 plays an important role in regulating the formation of TGP-43 nuclear bodies in stressed cells^[31].

To sum up, the C-terminal domain, post-translational modification and RNA of TDP-43 play an important role in the regulation of liquid-liquid phase separation and aggregation, so the regulation of liquid-liquid phase separation and aggregation of TDP-43 is a potential research direction.

4 Liquid-liquid phase separation and coacervation of Tau protein

Tauopathies are a group of neurodegenerative diseases caused by the misfolding of the microtubule-binding protein Tau and the formation of amyloid fibrils in neurons and glial cells, of which Alzheimer's disease is the most common tauopathy^[16].

It has been shown that Tau protein can easily undergo liquid-liquid phase separation to form aggregates in a physiologically crowded environment, and the concentration of Tau protein can be increased by more than 10-fold through phase separation^[11]. The liquid-liquid phase separation of Tau protein is sensitive to the concentration of salt ions, and decreasing the salt concentration can promote the phase separation, while increasing the salt concentration can inhibit the droplet formation. The sensitivity of phase separation to salt concentration suggests that electrostatic interactions are critical for Tau droplet formation^[43]. Truncation experiments indicate that electrostatic interactions between the positively charged domain and the negatively charged domain are the main driving force for the liquid-liquid phase separation of Tau protein to form condensates^[73]. Although the repeat region K18 of 4R Tau can also undergo liquid-liquid phase separation at very high concentrations, its phase separation is sensitive to 1,6-hexanediol rather than salt concentration, indicating that the main driving force for liquid-liquid phase separation of K18 is hydrophobic interaction rather than electrostatic interaction^[44]. At high salt concentrations, electrostatic interactions are screened and droplets formed by N-terminally truncated Tau can be dissolved by 1,6-hexanediol^[45]. Post-translational modifications can also regulate the liquid-liquid phase separation of Tau protein. The acetylation modification neutralizes the positive charge on lysine residues, thereby reducing the electrostatic attraction that drives intra- and intermolecular contacts, and thus, significantly inhibits Tau protein liquid-liquid phase separation to form condensates^[46]. Phosphorylation reverses the sensitivity of Tau protein liquid-liquid phase separation to salt concentration and 1,6-hexanediol. Liquid-liquid phase separation of phosphorylated Tau was insensitive to the increase of salt concentration, but sensitive to the addition of 1,6-hexanediol, indicating that phosphorylation modification may inhibit Tau liquid-liquid phase separation driven by electrostatic interactions, but promote Tau liquid-liquid phase separation driven by hydrophobic interactions^[11]. Taken together, these conclusions suggest that the liquid-liquid phase separation of Tau protein is driven by a complex combination of electrostatic and hydrophobic interactions.

Tau forms droplets with a variety of different RNA types, possibly because of their complex coacervation effects^[47]. The interaction of lysine and arginine residues with RNA is essential for the formation of complex condensed phases. Phosphorylation reduces the tendency of Tau protein to separate from RNA liquid-liquid phase, which may be caused by electrostatic repulsion between phosphorylated residues and negatively charged RNA. Acetylation also inhibits the liquid-liquid phase separation of Tau protein and RNA, presumably because of the loss of a positive charge on the lysine side chain^[48].

Metal ions play an important role in regulating Tau function. Rane et al. Found that Al³⁺ and Zn²⁺ enhance Tau liquid-liquid phase separation to form aggregates^[49]. Singh et al. Found that two cysteines in the microtubule-binding domain were essential for the formation of Tau droplets induced by Zn²⁺ in vitro^[50]. Metal ions may promote the liquid-liquid phase separation of Tau protein by changing its conformation to favor intermolecular electrostatic attraction or mediate intermolecular contacts^[49]. Recent studies in our laboratory have shown that zinc ions can promote the co-localization and interaction between Tau protein and stress granule marker G3BP1 in cells, thereby promoting the liquid-liquid phase separation of Tau protein, aggravating the mitochondrial damage caused by Tau aggregation and promoting Tau protein toxicity, revealing the molecular mechanism of Tau protein phase separation and cytotoxicity regulated by zinc ions^[51].

In addition, many studies have shown that cytokines and small molecules can regulate the liquid-liquid phase separation of Tau protein to form aggregates. Protein disulfide isomerase can directly interact with Tau and inhibit the formation of Tau droplets and fibers^[52]. Myricetin, a natural polyphenolic compound, can significantly slow down the liquid-liquid phase separation of full-length Tau protein, shift the equilibrium phase boundary to a higher protein concentration, and then inhibit Tau protein aggregation^[53]. Bis-ANS and similar compounds can bidirectionally regulate the liquid-liquid phase separation of Tau protein. Below the critical concentration, these small molecules can promote liquid-liquid phase separation, while above this concentration, the formation of droplets is inhibited^[41].

Although the structure of Tau protein droplets is difficult to determine, some progress has been made. Using double electron resonance spectroscopy, the scientists found that the average distance between the two ends of the PHF6^* region remained unchanged when Tau protein and RNA were subjected to liquid-liquid phase separation^[54]. However, the entire polypeptide chain of Tau protein exhibits a more extended conformation in the droplet state^[55]. The secondary structure of K18 was characterized by circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy, and it was found that liquid-liquid phase separation increased the content of β-structure and the tendency of β-hairpin conformation^[55]. Although the thioflavin T fluorescence of Tau protein droplets gradually increased with time, it was much weaker than the fluorescence intensity observed in the presence of heparin, so the β-structure content in droplets was much smaller than that in amyloid fibrils^[54].

In summary, the liquid-liquid phase separation of Tau protein is driven by both electrostatic and hydrophobic interactions. Post-translational modification, RNA, metal ions and molecular chaperones play an important role in the regulation of Tau protein liquid-liquid phase separation and aggregation, so the regulation of Tau protein liquid-liquid phase separation and aggregation is a potential research direction.

5 Discussion

In summary, low-complexity domains, RNAs, and molecular chaperones play an important role in regulating the liquid-liquid phase separation and aggregation of proteins associated with neurodegenerative diseases. Our laboratory has proposed that proteins and RNAs with low complexity domains form droplets by liquid phase condensation, then transform to the gel state, and finally form solid phase condensation-amyloid fibrils by solid phase condensation (Fig. 2). Therefore, elucidating the specific regulatory mechanisms of RNA and molecular chaperones in the phase separation of important disease-related proteins (prion protein, TDP-43 and Tau protein) will help to find ways to prevent and treat neurodegenerative diseases, and provide a theoretical basis for drug development.

6 Challenges and Prospects

Abnormal protein aggregation and fibrosis are the hallmark events in the pathogenesis of many neurodegenerative diseases. In recent years, the concept of liquid-liquid phase separation has become a new hot spot in the field of biology, and many proteins that form pathological aggregates can also undergo liquid-liquid phase separation under physiological conditions or in vitro.However, the relationship and causes of the two dynamic processes of protein aggregation and liquid-liquid phase separation are still unclear, and the related research is still in the embryonic stage, which can be predicted to be of great significance for us to understand the molecular mechanism of protein lesions and develop corresponding inhibitory means. It is worth noting that all studies of liquid-liquid phase separation of neurodegenerative disease-related proteins have been in vitro or in cells, and so far, no in vivo studies have shown a direct link between liquid-liquid phase separation of these proteins and neurodegenerative diseases, or that they occur in brain nerve cells. Finding experimental evidence for a direct link between liquid-liquid phase separation of proteins associated with neurodegenerative diseases and neurodegenerative diseases is both a challenge and an opportunity for researchers in this field. This article reviews the mechanism of protein aggregation in transmissible spongiform encephalopathies, TDP-43 proteinopathies and tauopathies.This paper focuses on how phase separation induces the formation of oligomers and amyloid fibrils from misfolded prion protein, TDP-43 and Tau protein in neurodegenerative diseases, and discusses the challenges and opportunities in the study of the relationship between protein aggregation and neurodegenerative diseases. It is known that the sequence composition, structure and motif of a protein itself are related to its potential to undergo liquid-liquid phase separation to form aggregates, and that the liquid-liquid phase separation of a protein can be affected by the intracellular environment, and can also be regulated by mutations and post-translational modifications.RNA, molecular chaperones and many small molecular ligands can regulate the liquid-liquid phase separation process, which may become potential therapeutic targets and provide new ideas for human to overcome neurodegenerative diseases.

References

Publishing order | Descend order by publishing year | Descend order by cited within

[1]	Palomo G M, Manfredi G. Brain Res., 2015, 1607: 36.

[2]	Weber S C, Brangwynne C P. Cell, 2012, 149(6): 1188.

[3]	Mitrea D M, Kriwacki R W. Cell Commun. Signal., 2016, 14: 1.

[4]	Boeynaems S, Alberti S, Fawzi N L, Mittag T, Polymenidou M, Rousseau F, Schymkowitz J, Shorter J, Wolozin B, van den Bosch L, Tompa P, Fuxreiter M. Trends Cell Biol., 2018, 28(6): 420.

[5]	Shin Y, Brangwynne C P. Science, 2017, 357(6357): eaaf4382.

[6]	Molliex A, Temirov J, Lee J H, Coughlin M, Kanagaraj A P, Kim H J, Mittag T, Taylor J P. Cell, 2015, 163(1): 123.

[7]	Wegmann 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 Y, Molliex A M, Hernandez-Vega A, Muller D, Hyman A A, Mandelkow E, Taylor J P, Hyman B T. EMBO J., 2018, 37(7): e98049.

[8]	Mathieu C, Pappu R V, Taylor J P. Science, 2020, 370(6512): 56.

[9]	Scheckel C, Aguzzi A. Nat. Rev. Genet., 2018, 19(7): 405.

[10]	Wang Y P, Mandelkow E. Nat. Rev. Neurosci., 2016, 17(1): 22.

[11]	Agarwal A, Mukhopadhyay S. J. Mol. Biol., 2022, 434(1): 167368.

[12]	Kostylev M A, Tuttle M D, Lee S, Klein L E, Takahashi H, Cox T O, Gunther E C, Zilm K W, Strittmatter S M. Mol. Cell, 2018, 72(3): 426.

[13]	Matos C O, Passos Y M, Amaral M J, Macedo B, Tempone M H, Bezerra O C L, Moraes M O, Almeida M S, Weber G, Missailidis S, Silva J L, Uversky V N, Pinheiro A S, Cordeiro Y. FASEB J., 2020, 34(1): 365.

[14]	Tange H, Ishibashi D, Nakagaki T, Taguchi Y, Kamatari Y O, Ozawa H, Nishida N. J. Biol. Chem., 2021, 296: 100367.

[15]	Agarwal A, Rai S K, Avni A, Mukhopadhyay S. PNAS, 2021, 118(45): e2100968118.

[16]	M Passos Y, J do Amaral M, C Ferreira N, Macedo B, Chaves J A P, de Oliveira V E, P B Gomes M, L Silva J, Cordeiro Y. Int. J. Biol. Macromol., 2021, 173: 34.

[17]	Agarwal A, Arora L, Rai S K, Avni A, Mukhopadhyay S. Nat. Commun., 2022, 13: 1154.

[18]	Li H R, Chiang W C, Chou P C, Wang W J, Huang J R. J. Biol. Chem., 2018, 293(16): 6090.

[19]	Hallegger M, Chakrabarti A M, Lee F C Y, Lee B L, Amalietti A G, Odeh H M, Copley K E, Rubien J D, Portz B, Kuret K, Huppertz I, Rau F, Patani R, Fawzi N L, Shorter J, Luscombe N M, Ule J. Cell, 2021, 184(18): 4680.

[20]	Pantoja-Uceda D, Stuani C, Laurents D V, McDermott A E, Buratti E, Mompeán M. PLoS Biol., 2021, 19(4): e3001198.

[21]	Wang A L, Conicella A E, Schmidt H B, Martin E W, Rhoads S N, Reeb A N, Nourse A, Ramirez Montero D, Ryan V H, Rohatgi R, Shewmaker F, Naik M T, Mittag T, Ayala Y M, Fawzi N L. EMBO J., 2018, 37(5): e97452.

[22]	Wang C, Duan Y, Duan G, Wang Q, Zhang K, Deng X, Qian B, Gu J, Ma Z, Zhang S, Guo L, Liu C, Fang Y. Mol. Cell, 2020, 79(3): 443.

[23]	Gasset-Rosa F, Lu S, Yu H Y, Chen C, Melamed Z, Guo L, Shorter J, da Cruz S, Cleveland D W. Neuron, 2019, 102(2): 339.

[24]

MacKenzie I R, Nicholson A M, Sarkar M, Messing J, Purice M D, Pottier C, Annu K, Baker M, Perkerson R B, Kurti A, Matchett B J, Mittag T, Temirov J, Hsiung G Y R, Krieger C, Murray M E, Kato M, Fryer J D, Petrucelli L, Zinman L, Weintraub S, Mesulam M, Keith J, Zivkovic S A, Hirsch-Reinshagen V, Roos R P, Züchner S, Graff-Radford N R, Petersen R C, Caselli R J, Wszolek Z K, Finger E, Lippa C, Lacomis D, Stewart H, Dickson D W, Kim H J, Rogaeva E, Bigio E, Boylan K B, Taylor J P, Rademakers R. Neuron, 2017, 95(4): 808.

[25]	Krainer G, Welsh T J, Joseph J A, Espinosa J R, Wittmann S, Sridhar A, Toprakcioglu Z, Gudiškyt?G, Czekalska M A, Arter W E, GuillÉn-Boixet J, Franzmann T M, Qamar S, George-Hyslop P S, Hyman A A, Collepardo-Guevara R, Alberti S, Knowles T P J. Nat. Commun., 2021, 12: 1085.

[26]	McGurk L, Gomes E, Guo L, Mojsilovic-Petrovic J, Tran V, Kalb R G, Shorter J, Bonini N M. Mol. Cell, 2018, 71(5): 703.

[27]	Fang M Y, Markmiller S, Vu A Q, Javaherian A, Dowdle W E, Jolivet P, Bushway P J, Castello N A, Baral A, Chan M Y, Linsley J W, Linsley D, Mercola M, Finkbeiner S, Lecuyer E, Lewcock J W, Yeo G W. Neuron, 2019, 103(5): 802.

[28]	Ambadipudi S, Biernat J, Riedel D, Mandelkow E, Zweckstetter M. Nat. Commun., 2017, 8: 275.

[29]	Ferreon J, Jain A, Choi K J, Tsoi P, MacKenzie K, Jung S, Ferreon A. Int. J. Mol. Sci., 2018, 19(5): 1360.

[30]	Lin Y, McCarty J, Rauch J N, Delaney K T, Kosik K S, Fredrickson G H, Shea J E, Han S. Elife, 2019, 8: e42571.

[31]	Ukmar-Godec T, Hutten S, Grieshop M P, Rezaei-Ghaleh N, Cima-Omori M S, Biernat J, Mandelkow E, Söding J, Dormann D, Zweckstetter M. Nat. Commun., 2019, 10: 2909.

[32]	Singh V, Xu L, Boyko S, Surewicz K, Surewicz W K. J. Biol. Chem., 2020, 295(18): 5850.

[33]	Gao Y Y, Zhong T, Wang L Q, Zhang N, Zeng Y, Hu J Y, Dang H B, Chen J, Liang Y. Int. J. Biol. Macromol., 2022, 209: 703.

[34]	Wang K, Liu J Q, Zhong T, Liu X L, Zeng Y, Qiao X H, Xie T, Chen Y Z, Gao Y Y, Tang B, Li J, Zhou J, Pang D W, Chen J, Chen C, Liang Y. J. Mol. Biol., 2020, 432(7): 2141.

[35]	Zhang X M, Lin Y X, Eschmann N A, Zhou H J, Rauch J N, Hernandez I, Guzman E, Kosik K S, Han S. PLoS Biol., 2017, 15(7): e2002183.

[36]	Majumdar A, Dogra P, Maity S, Mukhopadhyay S. J. Phys. Chem. Lett., 2019, 10(14): 3929.

[37]	Jonchhe S, Pan W, Pokhrel P, Mao H B. Angewandte Chemie Int. Ed., 2022, 61(23): e202113156.

[38]	Prasad A, Bharathi V, Sivalingam V, Girdhar A, Patel B K. Front. Mol. Neurosci., 2019, 12: 25.

[39]	Verdile V, de Paola E, Paronetto M P. Front. Genet., 2019, 10: 173.

[40]	Prusiner S B. PNAS, 1998, 95(23): 13363.

[41]	Prusiner S B. Trends Biochem. Sci., 1996, 21(12): 482.

[42]	Pan K M, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick R J, Cohen F E. PNAS, 1993, 90(23): 10962.

[43]	Diaz-Espinoza R, Soto C. Nat. Struct. Mol. Biol., 2012, 19(4): 370.

[44]	Wang L Q, Zhao K, Yuan H Y, Wang Q, Guan Z Y, Tao J, Li X N, Sun Y P, Yi C W, Chen J, Li D, Zhang D L, Yin P, Liu C, Liang Y. Nat. Struct. Mol. Biol., 2020, 27(6): 598.

[45]	Polymenidou M, Cleveland D W. Cell, 2011, 147(3): 498.

[46]	Wang L Q, Ma Y, Yuan H Y, Zhao K, Zhang M Y, Wang Q, Huang X, Xu W C, Dai B, Chen J, Li D, Zhang D, Wang Z, Zou L, Yin P, Liu C, Liang Y. Nat. Commun., 2022, 13(1): 3491.

[47]	Buratti E, Baralle F E. Trends Biochem. Sci., 2012, 37(6): 237.

[48]	Neumann M, Sampathu D M, Kwong L K, Truax A C, Micsenyi M C, Chou T T, Bruce J, Schuck T, Grossman M, Clark C M, McCluskey L F, Miller B L, Masliah E, MacKenzie I R, Feldman H, Feiden W, Kretzschmar H A, Trojanowski J Q, Lee V M Y. Science, 2006, 314(5796): 130.