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. 2019 Oct 7:2019:8130481.
doi: 10.1155/2019/8130481. eCollection 2019.

Pueraria lobata and Daidzein Reduce Cytotoxicity by Enhancing Ubiquitin-Proteasome System Function in SCA3-iPSC-Derived Neurons

I-Cheng Chen #  1 , Kuo-Hsuan Chang #  1 , Yi-Jing Chen  1 , Yi-Chun Chen  1 , Guey-Jen Lee-Chen  2 , Chiung-Mei Chen  1
Affiliations

Pueraria lobata and Daidzein Reduce Cytotoxicity by Enhancing Ubiquitin-Proteasome System Function in SCA3-iPSC-Derived Neurons

I-Cheng Chen et al. Oxid Med Cell Longev. .

Abstract

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion within the ATXN3/MJD1 gene. The expanded CAG repeats encode a polyglutamine (polyQ) tract at the C-terminus of the ATXN3 protein. ATXN3 containing expanded polyQ forms aggregates, leading to subsequent cellular dysfunctions including an impaired ubiquitin-proteasome system (UPS). To investigate the pathogenesis of SCA3 and develop potential therapeutic strategies, we established induced pluripotent stem cell (iPSC) lines from SCA3 patients (SCA3-iPSC). Neurons derived from SCA3-iPSCs formed aggregates that are positive to the polyQ marker 1C2. Treatment with the proteasome inhibitor, MG132, on SCA3-iPSC-derived neurons downregulated proteasome activity, increased production of radical oxygen species (ROS), and upregulated the cleaved caspase 3 level and caspase 3 activity. This increased susceptibility to the proteasome inhibitor can be rescued by a Chinese herbal medicine (CHM) extract NH037 (from Pueraria lobata) and its constituent daidzein via upregulating proteasome activity and reducing protein ubiquitination, oxidative stress, cleaved caspase 3 level, and caspase 3 activity. Our results successfully recapitulate the key phenotypes of the neurons derived from SCA3 patients, as well as indicate the potential of NH037 and daidzein in the treatment for SCA3 patients.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Characterization of SCA3 patient-specific-induced pluripotent stem cells (SCA3-iPSCs). (a) iPSC colonies obtained from reprogramming of normal controls and patients' fibroblasts. (b) Genotyping of iPSCs and fibroblasts (HF). PCR analysis showed SCA3 patients carrying expanded CAG repeats (66~68 repeats). (c) Immunofluorescent staining for the stem cell markers alkaline phosphatase (AP), OCT4 and TRA-1-81 in control- and SCA3-iPSCs. (d) RT-PCR analysis of exogenous and endogenous pluripotency markers (OCT4, KLF4, SOX2, and c-MYC genes) from iPSC cell lines. (e) Karyotyping analysis of SCA3-iPSCs. (f) Teratoma formed after subcutaneously injecting SCA3-iPSCs into nude mice. HE staining of teratomas showed endoderm, mesoderm, and ectoderm lineages. Scale bar: 100 μm.
Figure 2
Figure 2
Differentiation of neural progenitors and neurons from control- and SCA3-iPSCs. (a) Flow chart of neuronal differentiation from iPSCs. KO: Knockout medium; SB: SB431542. (b) Neural stem cells (NSCs) positive to NESTIN (green) and PAX6 (red) were generated following three weeks of differentiation from iPSCs. (c) Matured neurons expressing TUJ1 (red) were obtained following another two weeks of differentiation from NSCs. Scale bar: 50 μm. (d) Cell viability was determined by the trypan blue exclusion assay, and (e) caspase 3 activity was examined in the control- (NC-) and SCA3-iPSC-derived neurons. The caspase 3 activity in NC-iPSC-derived (NC-1 and NC-2) neurons was normalized as 100%. Each experiment for each sample was performed in triplicate.
Figure 3
Figure 3
PolyQ aggregation and proteasome activity of SCA3-iPSC-derived neurons. (a) Filter trap assay was conducted with 1C2 antibody staining to detect the accumulation of polyQ aggregates. (b) SCA3-iPSC-derived neurons showed 1C2 staining-positive aggregates (green) and the aggregates colocalized with p62 (red) (white arrow). Scale bar: 50 μm. (c) Proteasome activity assay was performed to analyze UPS function in iPSC-derived neurons. NC-iPSC-derived neurons (NC-1 and NC-2) were normalized as 100%. Each experiment for each sample was performed in triplicate.
Figure 4
Figure 4
Impairment of the ubiquitin-proteasome system (UPS) in SCA3-iPSC-derived neurons treated with MG132. (a) Proteasome activity of iPSC-derived neurons treated with various concentrations of MG132 (0~5 μM). Proteasome activity of untreated neurons was normalized as 100%. (b) 1C2-positive polyQ aggregates in 2 μM MG132-treated iPSC-derived neurons were detected by the filter trap assay. (c) Representative images of iPSC-derived neurons stained with 1C2 (green) and anti-TUJ1 (red). The white arrows in the magnified picture indicate 1C2-positive aggregates colocalized with TUJ1. (d) Representative images of iPSC-derived neurons stained with a cellular ROS-detecting reagent (green). The white arrows in the magnified picture indicate ROS detected in TUJ1-positive neurons. Scale bar: 50 μm. Each experiment for each sample was performed in triplicate. p values: MG132-treated vs. untreated, ∗∗p < 0.01 and ∗∗∗p < 0.001; SCA3-1 vs. NC-1 or SCA3-2 vs. NC-2, &p < 0.05, &&p < 0.01, and &&&p < 0.001.
Figure 5
Figure 5
Elevated cytotoxicity and caspase 3 expression/activity in MG132-treated iPSC-derived neurons. (a) Lactic dehydrogenase (LDH) release in culture media was measured to evaluate cytotoxicity. LDH level of untreated neurons was normalized as 100%. (b) Western blot analysis of the cleaved caspase 3 level (normalized to TUJ1 as the internal control). Cleaved caspase 3 level of 2 μM MG132-treated NC-iPSC-derived neurons (NC-1 and NC-2) was set as 100%. (c) Caspase 3 activity assay of NC- and SCA3-iPSC-derived neurons. Caspase 3 activity of untreated neurons was normalized as 100%. Each experiment for each sample was performed in triplicate. p values: MG132-treated vs. untreated, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; SCA3-1 vs. NC-1 or SCA3-2 vs. NC-2, &p < 0.05, &&p < 0.01, and &&&p < 0.001.
Figure 6
Figure 6
NH037 and daidzein promoted proteasome function and ameliorated oxidative stress in MG132-treated SCA3-iPSC-derived neurons. (a) Proteasome activity of iPSC-derived neurons treated with 2 μM MG132 alone or MG132 together with 1 mg/ml NH037 or 50 μM daidzein. The proteasome activity of untreated neurons was normalized as 100%. (b) Western blot analysis of ubiquitin. TUJ1 was used as the internal control. Ubiquitinated protein level of MG132-treated NC-iPSC-derived neurons (NC-1 and NC-2) was set as 100%. (c) Lipid peroxidation malondialdehyde (MDA) assay was conducted to evaluate oxidative stress. MDA level of untreated neurons was normalized as 100%. (d) Representative images of SCA3 neurons staining with TUJ1 (red) and cellular ROS detecting reagent (green) after treatment with 2 μM MG132, 1 mg/ml NH037, or 50 μM daidzein. Scale bar: 50 μm. Each experiment for each sample was performed in triplicate. p values: MG132-treated vs. untreated, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; SCA3-1 vs. NC-1 or SCA3-2 vs. NC-2, &p < 0.05; MG132/NH037 vs. MG132 alone or MG132/daidzein vs. MG132 alone, #p < 0.05, ##p < 0.01, and ###p < 0.001.
Figure 7
Figure 7
NH037 and daidzein reduced cytotoxicity and caspase 3 expression/activity in MG132-treated SCA3-iPSC-derived neurons. (a) LDH assay in iPSC-derived neurons treated with 2 μM MG132 alone or MG132 and 1 mg/ml NH037/50 μM daidzein (untreated neurons were normalized as 100%). (b) Western blot analysis of cleaved caspase 3 (normalized to TUJ1 as the internal control) in iPSC-derived neurons treated with 2 μM MG132 alone or MG132 and 1 mg/ml NH037/50 μM daidzein. The levels of cleaved caspase 3 from MG132-treated NC-iPSC-derived neurons (NC-1 and NC-2) were set as 100%. (c) Caspase 3 activity in iPSC-derived neurons treated with 2 μM MG132 alone or MG132 and 1 mg/ml NH037/50 μM daidzein. Untreated neurons were normalized as 100%. Each experiment for each sample was performed in triplicate. p values: MG132-treated vs. untreated, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; SCA3-1 vs. NC-1 or SCA3-2 vs. NC-2, &p < 0.05, &&p < 0.01, and &&&p < 0.001; MG132/NH037 vs. MG132 alone or MG132/daidzein vs. MG132 alone, #p < 0.05, ##p < 0.01.
Figure 8
Figure 8
Hypothesis of NH037 and daidzein targeting UPS function in SCA3-iPSC-derived neurons. In SCA3-iPSC-derived neurons, UPS function is impaired under environmental stress (such as MG132 exposure), and ubiquitinated proteins and polyQ-expanded ATXN3 protein are accumulated in cells. The resulting abnormal aggregation increased the production of oxidative stress and cytotoxicity. NH037 and daidzein promote protein degradation to reduce oxidative stress and cytotoxicity by improving proteasome activity in SCA3-iPSC-derived neurons.
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