Acetylation-induced TDP-43 pathology is suppressed by an HSF1-dependent chaperone program

Wang, Ping; Wander, Connor M.; Yuan, Chao; Bereman, Michael S.; Cohen, Todd J.

doi:10.1038/s41467-017-00088-4

Cited by 68 publications

(85 citation statements)

References 70 publications

(82 reference statements)

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“…Moreover, we identify K136 in the RRM1 as a potential regulatory site for TDP-43 RNA binding and splicing activity. Although we could confirm the stippled distribution and reduced CFTR splicing activity for the previously reported [K145Q]TDP-43 17,18 , the effects for K136 located directly within an essential RRM1 site appeared stronger in our experimental system. Acetylation can modulate the binding to RNA of other hnRNPs and therefore likely for TDP-43 as well 42,43 .…”

Section: Discussioncontrasting

confidence: 69%

“…2e,f), linking the formation of nuclear aggregates with TDP-43 loss of function. The previously described 17,18 K145Q mutant TDP-43 also showed some reduction of CFTR splice activity, although the effect was not as strong as for the K136 mutants expressed at comparably high protein levels (Fig. 2e).…”

Section: Resultsmentioning

confidence: 58%

“…In addition lysine modifications have been reported, including ubiquitinations at various residues 15 and sumoylation and acetylation of the RRM1 16,17 . The putative acetyl-mimic [K145Q]TDP-43 was distributed in a stippled manner in transfected cells, eventually recapitulating pathological phosphorylation and recruitment of ALS-related factors into TDP-43 aggregates 18 . In addition, [K145Q]TDP-43 had reduced CFTR splicing activity.…”

Section: Introductionmentioning

confidence: 99%

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Sirtuin-1 Sensitive Lysine-136 Acetylation Drives Phase Separation and Pathological Aggregation of TDP-43

Morato

Hans

Zweydorf

et al. 2020

Preprint

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The trans-activation response DNA-binding protein TDP-43 regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Confirming the results from site-directed mutagenesis, we succeeded to introduce authentic acetyl-lysine at the identified sites via amber suppression.[AcK84]TDP-43 showed cytoplasmic mislocalization and the aggregation propensity of [acK136]TDP-43 was confirmed. With newly developed antibodies, we found that the nuclear sirtuin SIRT1 can potently deacetylate [acK136]TDP-43. Moreover, SIRT1 reduced the aggregation propensity of [acK136]TDP-43. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting novel regulatory mechanisms for TDP-43 pathogenesis.the biochemical assay (compare Fig. 1d with Fig. 1b). Thus, acetylation of K84 partially reduced nuclear import of TDP-43 whereas the acetyl-mimic K79Q substitution did not show evident changes in nucleocytoplasmic distribution of TDP-43, likely because K79 is localized just outside the NLS, whereas K84 is at the core of the NLS 6,15 . The RRM1 mutants K136R and K136Q were predominantly localized in the nucleus, but a significant number of cells displayed a droplet-like pattern of nuclear aggregates (Fig. 1b,c). This droplet-like nuclear distribution was reminiscent of the RNA-binding deficient F147L/F149L mutant TDP-43 27 . For comparison, we generated the previously described 17,18 acetyl-mimic K145Q mutant in RRM1, which showed the expected stippled distribution (Fig. 1b). As K136 is in direct contact with bound nucleic acids 28,29 , we assume that modifications at K136 disrupt nucleic acid binding and therefore disengage TDP-43 from hnRNP complex localizations. Such dissociated K136-modified TDP-43 may be free to self-aggregate into inclusions. K136 mutant TDP-43 is pathologically ubiquitinated, phosphorylated, and insolubleWe examined if the K136 mutant TDP-43 inclusions in cell culture also showed pathological features described for human patients 4,5,13,30 . 6xHis-tagged TDP-43 variants were transiently transfected into HEK293E cells with stable knockdown of endogenous TDP-43 (sh TDP-43 ) 31 to minimize interference from the endogenous wtTDP-43. To assess protein solubility, RIPA-urea solubility fractionation assays were performed. While most of the 6xHis-tagged wtTDP-43 was RIPA-soluble, a larger portion of both K136R and K136Q TDP-43 mutants shifted into the RIPA-insoluble fraction (Fig. 2a). Although [F147L/F149L]TDP-43 formed similar intranuclear patches 27 , this designed RNA-...

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Section: Discussioncontrasting

confidence: 69%

Section: Resultsmentioning

confidence: 58%

Section: Introductionmentioning

confidence: 99%

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Sirtuin-1 Sensitive Lysine-136 Acetylation Drives Phase Separation and Pathological Aggregation of TDP-43

Morato

Hans

Zweydorf

et al. 2020

Preprint

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“…Acetylation of K145 and K192 increased aggregation of TDP-43 [143,144]. C-terminal fragmentation at D89 and D219 produced aggregation-prone TDP-43 fragments (residues 90-414 or residues 220-414, respectively) [145].…”

Section: Tar Dna-binding Protein 43 Ptms and Propensity For Aggregationmentioning

confidence: 99%

Do Post-Translational Modifications Influence Protein Aggregation in Neurodegenerative Diseases: A Systematic Review

Schaffert

Carter

2020

Brain Sciences

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The accumulation of abnormal protein aggregates represents a universal hallmark of neurodegenerative diseases (NDDs). Post-translational modifications (PTMs) regulate protein structure and function. Dysregulated PTMs may influence the propensity for protein aggregation in NDD-proteinopathies. To investigate this, we systematically reviewed the literature to evaluate effects of PTMs on aggregation propensity for major proteins linked to the pathogenesis and/or progression of NDDs. A search of PubMed, MEDLINE, EMBASE, and Web of Science Core Collection was conducted to retrieve studies that investigated an association between PTMs and protein aggregation in seven NDDs: Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), spinocerebellar ataxias, transmissible spongiform encephalopathy, and multiple sclerosis. Together, 1222 studies were identified, of which 69 met eligibility criteria. We identified that the following PTMs, in isolation or combination, potentially act as modulators of proteinopathy in NDDs: isoaspartate formation in Aβ, phosphorylation of Aβ or tau in AD; acetylation, 4-hydroxy-2-neonal modification, O-GlcNAcylation or phosphorylation of α-synuclein in PD; acetylation or phosphorylation of TAR DNA-binding protein-43 in ALS, and SUMOylation of superoxide dismutase-1 in ALS; and phosphorylation of huntingtin in HD. The potential pharmacological manipulation of these aggregation-modulating PTMs represents an as-yet untapped source of therapy to treat NDDs.

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“…Inclusion formation 47 has been strongly correlated with the death of neurons in neurodegenerative diseases (Braak, which are well-characterised molecular chaperones capable of stabilising and re-folding 69 misfolded proteins, with additional functions in trafficking damaged proteins to proteasomal or 70 autophagy-lysosome degradation pathways (Leak 2014). Heat shock proteins have previously 71 been shown to interact with disease-associated mature aggregates in vitro (Cox, Whiten et prone, acetylation-mimicking mutant of ALS-associated TAR DNA-binding protein-43 (TDP-83 43) (Wang, Wander et al 2017). Likewise, over-expression of HSF1 in a mutant superoxide 84 dismutase-1 (SOD1) mouse model of ALS, led to a 34% decrease in the level of insoluble 85 SOD1 H46R/H48Q in spinal cord tissue compared to controls (Lin, Simon et al 2013).…”

Section: Introduction 40mentioning

confidence: 99%

Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal-like cells

Gil

Cox

McAlary

et al. 2020

Preprint

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16Protein aggregation that results in the formation of inclusions is strongly correlated with 17 neuronal death and is a pathological hallmark common to many neurodegenerative diseases, 18including amyotrophic lateral sclerosis (ALS) and Huntington's disease. Cells are thought to 19 dramatically up-regulate the levels of heat shock proteins during periods of cellular stress via 20 induction of the heat shock response (HSR). Heat shock proteins are well-characterised 21 molecular chaperones that interact with aggregation-prone proteins to either stabilise, refold, 22 or traffic protein for degradation. The reason why heat shock proteins are unable to maintain 23 the solubility of particular proteins in neurodegenerative disease is unknown. We sought to 24 determine whether neurodegenerative disease-associated protein aggregates can induce the 25 HSR. Here, we generated a neuroblastoma cell line that expresses a fluorescent reporter 26 under conditions of HSR induction, for example heat shock. Using these cells, we show that 27 the HSR is not induced by exogenous treatment with aggregated forms of Parkinson's 28 disease-associated a-synuclein or the ALS-associated G93A mutant of superoxide 29 dismutase-1 (SOD1 G93A ). Furthermore, flow cytometric analysis revealed that intracellular 30 expression of SOD1 G93A or a pathogenic form of polyQ-expanded huntingtin (Htt 72Q ), similarly, 31 results in no or low induction of the HSR. In contrast, expression of a non-pathogenic but 32 aggregation-prone form of firefly luciferase (Fluc) did induce an HSR in a significantly greater 33 proportion of cells. Finally, we show that HSR induction is dependent on the intracellular levels 34 of the aggregation-prone proteins, but the pathogenic proteins (SOD1 G93A and Htt 72Q ) elicit a 35 significantly lower HSR compared to the non-pathogenic proteins (Fluc). These results 36 suggest that pathogenic proteins either evade detection or impair induction of the HSR in 37

show abstract

Acetylation-induced TDP-43 pathology is suppressed by an HSF1-dependent chaperone program

Cited by 68 publications

References 70 publications

Sirtuin-1 Sensitive Lysine-136 Acetylation Drives Phase Separation and Pathological Aggregation of TDP-43

Sirtuin-1 Sensitive Lysine-136 Acetylation Drives Phase Separation and Pathological Aggregation of TDP-43

Do Post-Translational Modifications Influence Protein Aggregation in Neurodegenerative Diseases: A Systematic Review

Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal-like cells

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