Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, Loss of Nuclear TDP-43, and Cell Death

Gasset-Rosa, F.; Lu, Shan; Yu, Haiyang; Chen, Cong; Melamed, Ze ev; Guo, Lin; Shorter, James; Cruz, Sandrine Da; Cleveland, Don W.

doi:10.1016/j.neuron.2019.02.038

Cited by 383 publications

(499 citation statements)

References 74 publications

(82 reference statements)

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“…However, in disease, heavily phosphorylated and ubiquitylated aggregates of TDP-43 accumulate cytoplasmically (20), accompanied by its nuclear clearance. We recently reported that TDP-43 naturally de-mixes in the nucleus, and shuttles to the cytoplasm in response to multiple stresses (21). Loss of nuclear TDP-43 causes usage of cryptic splice (22)(23)(24) and polyadenylation sites (24), dysregulating many important neuronal genes such as HDAC6 (25) and stathmin-2 (22,24).…”

Section: Mislocalization and Aggregation Of The Heterogeneous Nuclearmentioning

confidence: 99%

“…Recognizing earlier work demonstrating that endogenous TDP-43 naturally phase separates in several cell culture models and in the mouse nervous system (21) and that an RNA-binding deficient TDP-43 promotes phase separation and aggregation in multiple cellular models (27)(28)(29), we tested if interaction between RNA and TDP-43 regulates LLPS of intranuclear TDP-43. TDP-43 contains an N-terminal self-association domain (30,31), a C-terminal LCD, and two conserved RNA recognition motifs (RRM1 and RRM2) whose RNA-binding activity can be abolished by acetylation of two lysine residues (K145 in RRM1 and K192 in RRM2) (27).…”

Section: Rna Binding-deficient Tdp-43 De-mixes Into Ilsa In Physiologmentioning

confidence: 99%

“…We tested how iLSA containing RNA binding deficient TDP-43 affected behavior of wild type TDP-43. We started with a cell line (21) in which lentiviral transduction had been used to functionally replace endogenously expressed TDP-43 with a fluorescent, mRuby2-tagged, wild type TDP-43 (hereafter TDP-43 WT-mRuby ), with TDP-43 autoregulation (19, 34) maintaining overall TDP-43 level ( Fig. S2A-C).…”

Section: Ilsa From Rna Binding Deficient Tdp-43 Recruit Wild Type Tdp-43mentioning

confidence: 99%

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TDP-43 and HSP70 phase separate into anisotropic, intranuclear liquid spherical annuli

Gasior

et al. 2020

Preprint

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One Sentence Summary:Acetylation of TDP-43 drives its phase separation into spherical annuli that form a liquid-insidea-liquid-inside-a-liquid. AbstractThe RNA binding protein TDP-43 naturally phase separates within cell nuclei and forms cytoplasmic aggregates in age-related neurodegenerative diseases. Here we show that acetylation-mediated inhibition of TDP-43 binding to RNA produces co-de-mixing of acetylated and unmodified TDP-43 into symmetrical, intranuclear spherical annuli whose shells and cores have liquid properties. Shells are anisotropic, like liquid crystals. Consistent with our modelling predictions that annulus formation is driven by components with strong self-interactions but weak interaction with TDP-43, the major components of annuli cores are identified to be HSP70 family proteins, whose chaperone activity is required to maintain liquidity of the core. Proteasome inhibition, mimicking reduction in proteasome activity during aging, induces TDP-43-containing annuli in neurons in rodents. Thus, we identify that TDP-43 phase separation is regulated by acetylation, proteolysis, and ATPase-dependent chaperone activity of HSP70.A seminal discovery in the last decade has been recognition that large biological molecules, especially RNA binding proteins, can undergo liquid-liquid phase separation (LLPS), resembling oil droplets in vinegar. Under certain physical conditions, proteins, nucleic acids, or a mixture of both in a complex solution can form two phases, a condensed de-mixed phase and more dilute aqueous phase (1). Inside the cell, proteins and/or nucleic acids de-mix into a condensed phase to form membraneless organelles which have been proposed to promote biological functions (2).One membraneless organelle is the nucleolus, first described in the early 1800's and now recognized to be composed of proteins that undergo LLPS (3-5). Discovery that P-granules are de-mixed compartments with liquid behaviour brought widespread recognition to LLPS and its ability to mediate subcellular compartmentalization in a biological context (6). Phase separation has been also proposed for heterochromatin and RNA bodies (1, 6, 7). LLPS potentially underlies the operational principle governing formation of important organelles and structures, such as centrosomes, nuclear pore complexes, and super enhancers (8-10).Few mechanisms to modulate intracellular LLPS have been identified. Disease-causing mutations of proteins such as FUS (11) and HNRNPA2B1 (12) alter their LLPS behaviors in vitro, but the physiological or pathological relevance of their intracellular LLPS has not been fully elucidated. Random, multivalent interactions among intrinsically disordered, low complexity domains (LCDs) of proteins are a major driving force for LLPS in vitro. Oligomerization of other domains is thought to be required for LLPS in vivo (13). The inherent randomness of multivalent interactions (14) favors a model of disordered alignment -a conventional liquid phase in which molecules randomly move. The evidence is compelling that multi...

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Section: Mislocalization and Aggregation Of The Heterogeneous Nuclearmentioning

confidence: 99%

Section: Rna Binding-deficient Tdp-43 De-mixes Into Ilsa In Physiologmentioning

confidence: 99%

Section: Ilsa From Rna Binding Deficient Tdp-43 Recruit Wild Type Tdp-43mentioning

confidence: 99%

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TDP-43 and HSP70 phase separate into anisotropic, intranuclear liquid spherical annuli

Gasior

et al. 2020

Preprint

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“…Mechanistic details on the dynamics of such granules in TDP-43 pathophysiology remains unclear. However, many different proteins are known to interact with and partition into the phase separated droplets of TDP-43 (40,41). In FTLD and ALS, the aberrant LLPS of TDP-43 has emerged as a driving factor for the aggregation of the protein (40,42,43).…”

Section: Introductionmentioning

confidence: 99%

Granulins modulate liquid-liquid phase separation and aggregation of TDP-43 C-terminal domain

Bhopatkar

Uversky

Rangachari

2019

Preprint

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Tar DNA binding protein (TDP-43) has emerged as a key player in many neurodegenerative pathologies including frontotemporal lobar degeneration (FTLD) and amyotropic lateral sclerosis (ALS). Important hallmarks of FTLD and ALS are the toxic cytoplasmic inclusions of Cterminal fragments of TDP-43 (TDP-43CTD), which are formed upon proteolytic cleavage of fulllength TDP-43 in the nucleus and subsequent transport to the cytoplasm. TDP-43CTD is also known to form stress granules (SGs) by coacervating with RNA in cytoplasm under stress conditions and are believed to be involved in modulating the pathologies. Among other factors affecting these pathologies, the pleiotropic protein called progranulin (PGRN) has gained significant attention lately. The haploinsufficiency of PGRN, caused by autosomal dominant mutations in GRN gene, results in its loss-of-function linked to FTLD and ALS. But precisely how the protein contributes to the pathology remains unknown. Recently, cleavage to GRNs were observed to be a significant part of FTLD and ALS progression with specific GRNs exacerbating TDP-43-induced toxicity in C.elegans. In this report, we show that GRNs -3 and -5 directly interact with TDP-43CTD to modulate latter's aggregation or stress granule formation in disparate ways in vitro. These results constitute the first observation of direct interaction between GRNs and TDP-43 and suggest a mechanism by which the loss of PGRN function could lead to FTLD and ALS.

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“…Under conditions of cellular stress, both TDP-43 and FUS re-localize into cytoplasmic stress granules (SGs) [6,7], which are dynamic mRNA-protein assemblies implicated in regulating mRNA function [8,9]. Given this, and in light of ALS-associated mutations that generally reduce SG dynamics, SGs have been theorized to promote formation of TDP-43 and FUS aggregates that are observed in ALS patients [10,11], though recent studies suggest SG-independent aggregation mechanism also likely exist [12][13] [14]. Regardless, various studies indicate that TDP-43 and FUS aggregates, or simply excessive cytoplasmic localization of TDP-43 or FUS, may result in a toxic gain of function that leads to motor-neuron degeneration [15][16][17][18][19] [20,21].…”

Section: Introductionmentioning

confidence: 99%

Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

Liu¹,

Byrd²,

Pei³

et al. 2019

Preprint

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Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) are aggregation-prone RNA-binding proteins that in ALS can mis-localize to the cytoplasm of affected motor neuron cells, often forming cytoplasmic aggregates in the process. Such mis-localization and aggregation are implicated in ALS pathology, though the mechanisms of TDP-43 and FUS cytoplasmic toxicity remains unclear.Recently, we determined that the endocytic function aids turnover of TDP-43 and reduces TDP-43 toxicity. Here, we identified that Cdc48 and Ubx3, a Cdc48 co-factor implicated in endocytic function, regulates the turnover and toxicity of TDP-43 and FUS expressed in S. cerevisiae. Cdc48 physically interacts and co-localizes with TDP-43, as does VCP in ALS patient tissue. In yeast, FUS toxicity also depends strongly on endocytic function, but not autophagy under normal conditions. FUS expression also impairs endocytic function, as previously observed with TDP-43. Taken together, our data identifies a role for Cdc48/VCP and endocytosis function in regulating TDP-43 and FUS toxicity and turnover. Furthermore, endocytic dysfunction may be a common defect affecting cytoplasmic clearance of ALS aggregation-prone proteins and may represent a novel therapeutic target of promise. KeywordsCdc48, VCP, Endocytosis, TDP-43, FUS, ALS Abbreviations ALS -Amyotrophic Lateral Sclerosis CORVET -class C core vacuole/endosome tethering CHX -Cycloheximide EGFR -Epidermal growth factor receptor FTLD -Frontotemporal lobar dementia FUS -Fused in Sarcoma HOPS -homotypic fusion and vacuole protein sorting IBMPFD -Inclusion Body Myopathy with early onset Pagets disease and Frontotemporal Dementia RRM -RNA Recognition Motif SG -Stress granule TDP-43 -TAR DNA binding protein 43 VCP -Vasolin containing proteinUnder conditions of cellular stress, both TDP-43 and FUS re-localize into cytoplasmic stress granules (SGs) [6,7], which are dynamic mRNA-protein assemblies implicated in regulating mRNA function [8,9]. Given this, and in light of ALS-associated mutations that generally reduce SG dynamics, SGs have been theorized to promote formation of TDP-43 and FUS aggregates that are observed in ALS patients [10,11], though recent studies suggest SG-independent aggregation mechanism also likely exist [12][13] [14]. Regardless, various studies indicate that TDP-43 and FUS aggregates, or simply excessive cytoplasmic localization of TDP-43 or FUS, may result in a toxic gain of function that leads to motor-neuron degeneration [15][16][17][18][19] [20,21]. While the mechanism of such toxicity remains unclear, preventing aggregation and/or enhancing clearance of cytoplasmic TDP-43 and FUS is of considerable therapeutic interest. This approach has shown promise in various ALS models where preventing SG assembly or upregulating cytoplasmic protein turnover has been tested [22][23][24][25][26].Recently, we and others demonstrated that under normal growth conditions, TDP-43 toxicity, aggregation and prote...

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Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, Loss of Nuclear TDP-43, and Cell Death

Cited by 383 publications

References 74 publications

TDP-43 and HSP70 phase separate into anisotropic, intranuclear liquid spherical annuli

TDP-43 and HSP70 phase separate into anisotropic, intranuclear liquid spherical annuli

Granulins modulate liquid-liquid phase separation and aggregation of TDP-43 C-terminal domain

Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

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