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. 2012;7(8):e42277.
doi: 10.1371/journal.pone.0042277. Epub 2012 Aug 3.

Inhibition of TDP-43 accumulation by bis(thiosemicarbazonato)-copper complexes

Sarah J Parker  1 , Jodi Meyerowitz, Janine L James, Jeffrey R Liddell, Takashi Nonaka, Masato Hasegawa, Katja M Kanninen, SinChun Lim, Brett M Paterson, Paul S Donnelly, Peter J Crouch, Anthony R White
Affiliations

Inhibition of TDP-43 accumulation by bis(thiosemicarbazonato)-copper complexes

Sarah J Parker et al. PLoS One. 2012.

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron disease with no effective long-term treatment options. Recently, TDP-43 has been identified as a key protein in the pathogenesis of some cases of ALS. Although the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has been shown to accumulate in RNA stress granules (SGs) in cell models and in spinal cord tissue from ALS patients. The SG association may be an early pathological change to TDP-43 metabolism and as such a potential target for therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of mitochondrial activity can be inhibited by modulation of cellular kinase activity. We have also found that treatment of cells and animal models of neurodegeneration, including an ALS model, with bioavailable bis(thiosemicarbazonato)copper(II) complexes (Cu(II)(btsc)s) can modulate kinase activity and induce neuroprotective effects. In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the Cu(II)(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The Cu(II)(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either Cu(II)(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. Cu(II)(atsm) treatment or ERK inhibition also prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a link between prolonged ERK activation and abnormal ubiquitin metabolism in paraquat stress and inhibition by Cu. Moreover, Cu(II)(atsm) reduced accumulation of C-terminal (219-414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that Cu(II)(btsc) complexes could potentially be developed as a neuroprotective agent to modulate neuronal kinase function and inhibit TDP-43 aggregation. Further studies in TDP-43 animal models are warranted.

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Conflict of interest statement

Competing Interests: ARW is currently a PLoS ONE Editorial Board Member. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Effect of CuII(atsm) and CuII(gtsm) on TDP-43 and HuR localization in SY5Y cells.
Cells were exposed overnight with 1 mM paraquat in the presence or absence of 1 µM CuII(atsm) or 50 nM CuII(gtsm) and TDP-43 and HuR localization was examined by immunofluorescence. A–D: untreated, E–H: paraquat, I–L: paraquat and CuII(atsm), M–P: paraquat and CuII(gtsm). Green  =  TDP-43, red  =  HuR, blue  =  DAPI. Bottom panels  =  merged images of TDP-43 and HuR above. Arrows indicate TDP-43 and HuR SGs. Bar  = 10 µm.
Figure 2
Figure 2. Effect of CuII(btsc)s and metals on TDP-43 and HuR SGs in SY5Y cells.
Cells were treated overnight with 1 mM paraquat in the presence or absence of 1 µM CuII(atsm), 1 and 100 µM CuCl2 and 100 µM FeCl2. A–B: untreated, C–D: paraquat, E–F: paraquat and CuII(atsm), G–H: paraquat and 1 µM CuCl2, I–J: paraquat and 100 µM CuCl2, K–L: paraquat and 100 µM FeCl2. Green  =  TDP-43, blue  =  DAPI. Arrows indicate SGs. Bar  = 10 µm. M: SGs (TDP-43 or HuR-positive) per cell after overnight treatment with 1 mM paraquat and co-treatment with CuII(atsm), CuII(gtsm), CuCl2 (1 and 100 µM) or FeCl2. **p<0.01 compared to paraquat treatment.
Figure 3
Figure 3. Effect of CuII(atsm) on SGs in HeLa cells induced by sodium arsenite.
Cells were treated for 1 hr with 500 µM sodium arsenite in the presence or absence of 1 µM CuII(atsm) (1 hr) and TDP-43 and HuR were examined by immunofluoresence. A–C: untreated, D–F: sodium arsenite treated, G–I: sodium arsenite and CuII(atsm). Green  =  TDP-43, red  =  HuR, blue  =  DAPI. Arrows indicate TDP-43 and HuR SGs. Bar  = 10 µm. J: SGs (TDP-43 or HuR-positive) per cell after 1 hr treatment with 500 µM sodium arsenite and co-treatment with 1 µM CuII(atsm).
Figure 4
Figure 4. Effect of CuII(atsm) on TDP-43, HuR and kinase phosphorylation.
Graphs show densitometric analysis of altered protein expression or phosphorylation from two-three separate experiments relative to loading controls. A: Cells were treated with 1 mM paraquat overnight in the presence or absence of 20 µM SP600125, 1 µM CuII(atsm) or 1 µM CuCl2 and immunoblotted for TDP-43 or HuR. B–C: Cells were treated for 2 hr with paraquat in the presence or absence of 1 µM CuII(atsm), 1 µM CuCl2 or 10 µM PD98059 (ERK inhibitor). Cells were immunoblotted for phospho-ERK (p-ERK), total ERK, phospho-JNK (p-JNK) and total JNK. D–E: Cells were treated overnight with paraquat in the presence or absence of 1 µM CuII(atsm), 1 µM CuCl2 or 10 µM PD98059 (ERK inhibitor). Cells were immunoblotted for phospho-ERK (p-ERK), total ERK, phospho-JNK (p-JNK) and total JNK. F: Effect of CuCl2 on ERK and JNK phosphorylation. Cells were treated overnight with 1 mM paraquat or paraquat plus 100 µM CuCl2 and cells were examined for expression ERK and JNK phosphorylation. G: HeLa cells were treated for 1 hr with 500 µM sodium arsenite in presence or absence of 1 µM CuII(atsm). Cells were immunoblotted for phospho-ERK (p-ERK), total ERK, phospho-JNK (p-JNK) and total JNK. *p<0.05 compared to untreated control. **p<0.05 compared to treatment alone.
Figure 5
Figure 5. Effect of CuII(atsm) and kinase inhibitors on TDP-43 and HuR-positive SG formation.
Cells were treated overnight with 1 mM paraquat in the presence or absence of 1 µM CuII(atsm), 10 µM PD98059 or 20 µM SP600125 and examined for TDP-43 and HuR by immunofluorescence. A–C: untreated, D–F: paraquat, G–I: paraquat and CuII(atsm), J–L: paraquat and PD98059, M–O: paraquat and SP600125. Green  =  TDP-43, red  =  HuR, blue  =  DAPI. Arrows indicate TDP-43 and HuR SGs. Bar  = 10 µm. P: SGs (TDP-43 or HuR-positive) per cell after overnight treatment with 1 mM paraquat and co-treatment with CuII(atsm) (1 µM), PD98095 or SP600125. **p<0.01 compared to paraquat treatment.
Figure 6
Figure 6. Effect of CuII(atsm) on paraquat-mediated changes to ubiquitin.
Cells were treated with 1 mM paraquat overnight in the presence or absence of 1 µM CuII(atsm), 10 µM PD98059 or 100 µM CuCl2. Cells were also exposed overnight to 75 µM rotenone. Cells were examined for TDP-43 and ubiquitin by immunofluorescence. A–C: untreated, D–F: paraquat, G–I: rotenone, J–L: paraquat and CuII(atsm), M–O: paraquat and PD98059 (ERK inhibitor), P–R: paraquat and CuCl2. Green  =  TDP-43, red  =  ubiquitin, blue  =  DAPI. Arrowheads indicate ubiquitin aggregation. Bar  = 10 µm. S: Percentage of cells containing ubiquitin aggregates after overnight treatment with 1 mM paraquat, 75 µM rotenone, paraquat plus 1 µM CuII(atsm), paraquat plus PD98059 or paraquat plus 100 µM CuCl2. **p<0.01 compared to paraquat treatment.
Figure 7
Figure 7. Effect of CuII(btsc)s on paraquat toxicity.
A: Cells were treated overnight with 1 or 10 mM paraquat in the presence or absence of 1 µM CuII(atsm), 50 nM CuII(gtsm) or 100 µM CuCl2 and cell death was measured with the LDH assay. *p<0.01 compared to untreated cells. **p<0.01 compared to 10 mM paraquat alone. B: In-gel SOD activity assay. Cells were treated with 1 mM paraquat overnight alone or in the presence of 1 µM CuII(atsm), 50 nM CuII(gtsm) or 1 µM CuCl2. CuII(atsm), CuII(gtsm) and CuCl2 were also added at the same concentration to cultures without paraquat. Cell lysates were analyzed for SOD activity using a SOD zymography gel assay. The band representing SOD activity was analyzed by densitometry and revealed no significant change compared to untreated control.
Figure 8
Figure 8. Effect of CuII(atsm) on aggregation of CTF-TDP-43 219–414.
Cells were transfected with WT TDP-43-GFP (full length) or CTF-TDP-43 219–414-GFP and formation of TDP-43 aggregates was examined after 48 hr. A–C: Aggregates of TDP-43 were not observed in cells transfected with WT-TDP-43. D–F: Upon transfection with CTF-TDP-43 219–414, cells revealed widespread formation of cytosolic aggregates after 48 hr. G–I: Addition of CuII(atsm) (1 µM) to cells at 24 hr resulted in a reduction in frequency of cells expressing CTF-TDP-43 219–414 aggregates at 48 hr. J–L: Addition of CuCl2 (1 µM) to cells at 24 hr resulted in no significant change to the frequency of cells expressing CTF-TDP-43 219–414 aggregates at 48 hr. C, F, I and L represent merged images of TDP and DAPI. Arrows indicate TDP-43 aggregates. Green  =  TDP-43, blue  =  DAPI. Bar  = 10 µm.
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References

    1. King AE, Dickson TC, Blizzard CA, Woodhouse A, Foster SS, et al. (2011) Neuron-glia interactions underlie ALS-like axonal cytoskeletal pathology. Neurobiol Aging 32: 459–69. - PubMed
    1. Bowling AC, Schulz JB, Brown RHJ, Beal MF (1993) Superoxide dismutase activity, oxidative damage, and mitochondrial energy metabolism in familial and sporadic amyotrophic lateral sclerosis. J Neurochem 61: 2322–2325. - PubMed
    1. Rosen DR (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 364: 362. - PubMed
    1. Barber SC, Shaw PJ (2010) Oxidative stress in ALS: key role in motor neuron injury and therapeutic target. Free Radic Biol Med 48: 629–641. - PubMed
    1. Swarup V, Julien JP (2010) ALS pathogenesis: Recent insights from genetics and mouse models. Prog Neuropsychopharmacol Biol Psychiatry 35: 363–369. - PubMed

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This work was supported by funding from the National Health and Medical Research Council of Australia and Australian Research Council (ARC). ARW is a recipient of an ARC Future Fellowship Award. PJC is recipient of a C. R. Roper Fellowship. KMK was supported by Sigrid Juselius Foundation, Finland. JLJ was supported by a Rotary Health Scholarship. The authors would also like to thank the Motor Neuron Disease Research Institute of Australia, Bethlehem Griffiths Research Foundation and CASS Foundation for the kind support of this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.