Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

Abstract

Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress–induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R–like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal–regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential. Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress–induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R–like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal–regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential. Frontotemporal lobar degeneration (FTLD) 2The abbreviations used are: FTLDfrontotemporal lobar degenerationBafbafilomycinCFTRcystic fibrosis transmembrane conductance regulatoreIFeukaryotic initiation factorERKextracellular signal–regulated kinaseFUSfused-in-sarcomaGAPDHglyceraldehyde 3-phosphate dehydrogenaseGSK3βglycogen synthase kinase 3βHDAC6histone deacetylase 6HEKhuman embryonic kidneyHMWhigher molecular weight smearHRPhorseradish peroxidaseJNKc-Jun N-terminal kinaseMAPKmitogen-activated protein kinaseNACN-acetylcysteinePKRprotein kinase RPERKPKR-like endoplasmic reticulum kinaseRIPAradioimmunoprecipitation assaySGstress granuleSKARribosomal subunit protein S6 kinase 1 Aly/REF-like targetTARtransactive response elementTDP-43TAR DNA–binding protein of 43 kDaRBPRNA-binding proteinERendoplasmic reticulumHRIheme-regulated inhibitormTORmechanistic target of rapamycinTIA-1T-cell–restricted intracellular antigen-1TIARTIA-1–related proteinp70S6KS6 protein kinaseUBPYubiquitin isopeptidase YWBWestern blottingDMEMDulbecco's modified Eagle's mediumMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseInhinhibitorNi-NTAnickel-nitrilotriacetic acidBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. and ALS are human neurodegenerative diseases causing symptoms of dementia and motoneuron degeneration, respectively. Neuropathological inclusions containing TAR DNA–binding protein of 43 kDa (TDP-43) occur in most cases, whereas a subset of these diseases is characterized by the protein fused-in-sarcoma (FUS). Moreover, mutations in the genes encoding TDP-43 and FUS are linked to ALS and rarely also to FTLD (1Mackenzie I.R. Rademakers R. Neumann M. TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia.Lancet Neurol. 2010; 9 (20864052): 995-100710.1016/S1474-4422(10)70195-2Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). It was evident from the beginning that TDP-43 is pathologically modified. Disease-associated modifications of TDP-43 include protein insolubility, ubiquitylation, phosphorylation, and proteolytic processing (2Neumann 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. 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Chem. 1996; 271 (8663194): 16586-1659010.1074/jbc.271.28.16586Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). We investigated pathological modifications of TDP-43 under SG-inducing conditions. Treatment of HEK293E cells with arsenite and sorbitol caused particularly strong TDP-43 ubiquitylation and insolubility and induced functional impairment of TDP-43. Interestingly, SG suppression did not prevent TDP-43 ubiquitylation and shifts into more insoluble fractions. Arsenite affected TDP-43 via oxidative stress, whereas sorbitol apparently stimulated novel pathways leading to TDP-43 ubiquitylation and insolubility. Importantly, TDP-43 accumulation within SGs was not a prerequisite for early steps of TDP-43 pathogenesis. To investigate stress-induced changes of potentially pathogenic TDP-43 polyubiquitylation, insolubility, and aggregate formation, we exposed human embryonic kidney HEK293E cells to various stress conditions and analyzed these modifications of TDP-43 by His6-ubiquitin pulldown, solubility assay, and immunofluorescence. As reported before (27Hans F. Fiesel F.C. Strong J.C. Jäckel S. Rasse T.M. Geisler S. Springer W. Schulz J.B. Voigt A. Kahle P.J. UBE2E ubiquitin-conjugating enzymes and ubiquitin isopeptidase Y regulate TDP-43 protein ubiquitination.J. Biol. Chem. 2014; 289 (24825905): 19164-1917910.1074/jbc.M114.561704Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 28Hans F. Eckert M. von Zweydorf F. Gloeckner C.J. Kahle P.J. Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43).J. Biol. Chem. 2018; 293 (30120199): 16083-1609910.1074/jbc.RA118.003440Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), the proteasome inhibitor MG-132 but not the autophagy inhibitor bafilomycin (Baf) A1 promoted ubiquitylation of FLAG-tagged TDP-43 in HEK293E cells (Fig. 1, A (right) and B). We added several stressors that had been implicated with TDP-43 mismetabolism, namely hyperosmolar concentrations of sorbitol (8Dewey C.M. Cenik B. Sephton C.F. Dries D.R. Mayer 3rd, P. Good S.K. Johnson B.A. Herz J. Yu G. TDP-43 is directed to stress granules by sorbitol, a novel physiological osmotic and oxidative stressor.Mol. Cell. Biol. 2011; 31 (21173160): 1098-110810.1128/MCB.01279-10Crossref PubMed Scopus (240) Google Scholar), heat shock, oxidative stress mediated by sodium arsenite or hydrogen peroxide (H2O2), and the ER stress inducer thapsigargin (9McDonald K.K. Aulas A. Destroismaisons L. Pickles S. Beleac E. Camu W. Rouleau G.A. Vande Velde C. TAR DNA-binding protein 43 (TDP-43) regulates stress granule dynamics via differential regulation of G3BP and TIA-1.Hum. Mol. Genet. 2011; 20 (21257637): 1400-141010.1093/hmg/ddr021Crossref PubMed Scopus (275) Google Scholar). These treatments can activate cellular stress responses involving the formation of SGs, which was suggested to be an important step in the development of TDP-43 and FUS inclusions in FTLD and ALS (17Bentmann E. Haass C. Dormann D. Stress granules in neurodegeneration—lessons learnt from TAR DNA binding protein of 43 kDa and fused in sarcoma.FEBS J. 2013; 280 (23587065): 4348-437010.1111/febs.12287Crossref PubMed Scopus (135) Google Scholar). We found that treatment with 0.4 m sorbitol most strongly induced ubiquitylation of TDP-43 (Fig. 1, A and B). Thermal stress mediated by 30-min temperature elevation to 43 °C also robustly caused TDP-43 ubiquitylation. Oxidative stress induced by treatment with 250 μm sodium arsenite also caused robust TDP-43 ubiquitylation, but treatment with 500 μm H2O2 failed in this experiment. Thapsigargin (1 μm) also caused no TDP-43 ubiquitylation. As reported previously (27Hans F. Fiesel F.C. Strong J.C. Jäckel S. Rasse T.M. Geisler S. Springer W. Schulz J.B. Voigt A. Kahle P.J. UBE2E ubiquitin-conjugating enzymes and ubiquitin isopeptidase Y regulate TDP-43 protein ubiquitination.J. Biol. Chem. 2014; 289 (24825905): 19164-1917910.1074/jbc.M114.561704Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 28Hans F. Eckert M. von Zweydorf F. Gloeckner C.J. Kahle P.J. Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43).J. Biol. Chem. 2018; 293 (30120199): 16083-1609910.1074/jbc.RA118.003440Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), proteasome inhibition with MG-132 not only enhanced TDP-43 ubiquitylation, but also shifted TDP-43 into the RIPA-insoluble urea fraction. The strongest TDP-43 ubiquitylation inducers (Fig. 1, A and B), sorbitol, arsenite, and heat shock shifted a relatively large portion of endogenous TDP-43 into the insoluble (urea) fraction (Fig. 1, C and D). The effect was even stronger than for MG-132 (Fig. 1D), consistent with enhanced TDP-43 ubiquitylation (Fig. 1A). We confirmed successful treatments by detection of various downstream (phosphorylation) targets (see Fig. 1C and the figure legend for further details). Interestingly, osmotic stress reduced mTOR phosphorylation and phosphorylation of ribosomal S6 protein kinase (p70S6K), consistent with translation-blocking SG signaling. At the same time, membrane-bound LC3B II accumulated. Thus, under these conditions, reduced mTOR signaling might initiate autophagy but with impaired autophagic flux. Immunofluorescence microscopy using two different SG markers (eIF3η and TIAR) did not reveal any SG formation after treatment with MG-132 and Baf A1, and TDP-43 remained mostly nuclear under these conditions (Fig. 2). H2O2 failed in our hands. Short exposure to the ER stressor thapsigargin caused cellular reactions, as evidenced by ERK1/2 and p70S6K phosphorylations (Fig. 1B), but did not effectively lead to SG formation (Fig. 2). As expected, sorbitol and arsenite treatments caused most prominent SG formation, as well as translocation of a considerable portion of TDP-43 into the cytosol, where it co-localized with SGs (Fig. 2). Strong induction of SG signaling by arsenite and sorbitol exposure was confirmed by detection of eIF2α phosphorylation (Fig. 1B). Heat shock caused quite strong TDP-43 ubiquitylation and insolubility (Fig. 1), although under these conditions no SG formation was visible (Fig. 2). SG signaling was not detected after 30-min heat shock; in fact, p70S6K phosphorylation was even enhanced (Fig. 1B). It appears that such a short heat shock does not induce global protein-misfolding stress in HEK293E cells, but the aggregation-prone TDP-43 may be affected even under limiting thermal stress conditions. Thus, although SG-forming conditions after treatment with arsenite and sorbitol cause very strong TDP-43 ubiquitylation and insolubility, TDP-43 recruitment into SGs is not a universal prerequisite for such TDP-43 modifications, which can happen after brief heat shock even in the absence of SGs. Enhanced TDP-43 ubiquitylation and insolubility could impair its functions by reducing the amount of active TDP-43 molecules. TDP-43 regulates many RNA-processing steps, including pre-mRNA splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) (29Buratti E. Dörk T. Zuccato E. Pagani F. Romano M. Baralle F.E. Nuclear factor TDP-43 and SR proteins promote in vitroin vivo CFTR exon 9 skipping.EMBO J. 2001; 20 (11285240): 1774-178410.1093/emboj/20.7.1774Crossref PubMed Scopus (499) Google Scholar) and the ribosomal subunit protein S6 kinase 1 Aly/REF-like target (SKAR) (29Buratti E. Dörk T. Zuccato E. Pagani F. Romano M. Baralle F.E. Nuclear factor TDP-43 and SR proteins promote in vitroin vivo CFTR exon 9 skipping.EMBO J. 2001; 20 (11285240): 1774-178410.1093/emboj/20.7.1774Crossref PubMed Scopus (499) Google Scholar, 30Fiesel F.C. Weber S.S. Supper J. Zell A. Kahle P.J. TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR.Nucleic Acids Res. 2012; 40 (22121224): 2668-268210.1093/nar/gkr1082Crossref PubMed Scopus (71) Google Scholar). TDP-43 also promotes histone deacetylase 6 (HDAC6) mRNA stability (31Fiesel F.C. Voigt A. Weber S.S. Van den Haute C. Waldenmaier A. Görner K. Walter M. Anderson M.L. Kern J.V. Rasse T.M. Schmidt T. Springer W. Kirchner R. Bonin M. Neumann M. Baekelandt V. Alunni-Fabbroni M. Schulz J.B. Kahle P.J. Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6.EMBO J. 2010; 29 (19910924): 209-22110.1038/emboj.2009.324Crossref PubMed Scopus (184) Google Scholar). We assessed whether the two strongest TDP-43–modifying stressors sorbitol and sodium arsenite could impair these TDP-43 functions. For comparison, the same stressors were applied to cells stably depleted of TDP-43 by RNAi. Cells were transfected with a CFTR splice reporter minigene (29Buratti E. Dörk T. Zuccato E. Pagani F. Romano M. Baralle F.E. Nuclear factor TDP-43 and SR proteins promote in vitroin vivo CFTR exon 9 skipping.EMBO J. 2001; 20 (11285240): 1774-178410.1093/emboj/20.7.1774Crossref PubMed Scopus (499) Google Scholar), and as expected under control conditions, TDP-43 promoted strong exon 9 skipping (Fig. 3). Arsenite treatment for 1–3 h led to a small accumulation of the unspliced exon 9 included product and thus resulted in a significant reduction of the splicing ratio (Fig. 3). The same effect was seen for the endogenous splice target SKAR. Arsenite treatment for 1–3 h caused an accumulation of the mis-spliced exon-skipped SKAR β isoform, resulting in significant reduction of the TDP-43–dependent splice ratio (Fig. 3). For sorbitol treatments, the trends were less significant, where we only observed impaired SKAR splicing. The arsenite- and sorbitol-induced effects were not confirmed for protein levels of SKAR α and β, likely because only short time courses could be reliably followed. Prolonged stress may be necessary for mis-spliced mRNA and protein turnover to reach saturation, but the cells did not tolerate well longer exposure times to sorbitol or arsenite. The HDAC6 mRNA levels were reduced in cells with stable TDP-43 shRNA expression (Fig. 3), as expected. Surprisingly, sorbitol and arsenite treatment did not diminish HDAC6 expression, but actually enhanced the levels of HDAC6 mRNA and, to a lesser extent, also HDAC6 protein levels (Fig. 3). HDAC6 induction occurred both in WT cells and in TDP-43 knockdown cells. Thus, TDP-43–independent pathways regulating HDAC6 expression must exist in our experimental system. Therefore, HDAC6 does not serve as a surrogate marker of TDP-43 activity under these conditions. Nevertheless, the observed CFTR and SKAR mis-splicing (see above) is indicative of some loss of TDP-43 activity under stress conditions that lead to ubiquitinylation and insolubility of a fraction of TDP-43 protein. The SG inducers sorbitol and arsenite very strongly promoted His6-ubiquitylation of FLAG-tagged TDP-43 in transfected HEK293E cells. In contrast, no HMW ubiquitylation was detected for 3xFLAG-tagged FUS in HEK293E cells treated with sorbitol or sodium arsenite (Fig. S1A), although endogenous FUS was efficiently recruited to SGs upon sorbitol exposure (Fig. S1B). Arsenite-induced SG translocation of FUS was barely detectable. Thus, the stress-induced ubiquitylations investigated here and our previous studies (27Hans F. Fiesel F.C. Strong J.C. Jäckel S. Rass