Additional file 1: of Molecular pathomechanisms and cell-type-specific disease phenotypes of MELAS caused by mutant mitochondrial tRNATrp

Supplementary Figures and Tables. (DOC 4252 kb) Figure S1. Comparison of mitochondrial tRNATrp stability between wild-type and m.5541C > T mutant (related to Fig. 1). Figure S2. Protein modeling and amino acid sequences of each mtDNA-encoded CIV subunit (related to Fig. 2). Figure S3. Generation of disease-relevant iPSCs carrying all mutant mitochondrial tRNATrp (related to Fig. 3). Figure S4. Mutant mitochondrial tRNATrp strongly impairs neuronal maturation (related to Fig. 4). Table S1. mtDNA sequence variants in this patient. Table S2. Primer list. Table S3. TaqMan probe list

Interleukin-1beta (IL-1β)-induced Notch ligand Jagged1 suppresses mitogenic action of IL-1β on human dystrophic myogenic cells

<div><p>Duchenne muscular dystrophy (DMD) is a severe X-linked recessive muscle disorder caused by mutations in the dystrophin gene. Nonetheless, secondary processes involving perturbation of muscle regeneration probably exacerbate disease progression, resulting in the fatal loss of muscle in DMD patients. A dysfunction of undifferentiated myogenic cells is the most likely cause for the reduction of regenerative capacity of muscle. To clarify molecular mechanisms in perturbation of the regenerative capacity of DMD muscle, we have established several NCAM (CD56)-positive immortalized human dystrophic and non-dystrophic myogenic cell lines from DMD and healthy muscles. A pro-inflammatory cytokine, IL-1β, promoted cell cycle progression of non-dystrophic myogenic cells but not DMD myogenic cells. In contrast, IL-1β upregulated the Notch ligand Jagged1 gene in DMD myogenic cells but not in non-dystrophic myogenic cells. Knockdown of Jagged1 in DMD myogenic cells restored the IL-1β-promoted cell cycle progression. Conversely, enforced expression of Jagged1-blocked IL-1β promoted proliferation of non-dystrophic myogenic cells. In addition, IL-1β prevented myogenic differentiation of DMD myogenic cells depending on Jagged1 but not of non-dystrophic myogenic cells. These results demonstrate that Jagged1 induced by IL-1β in DMD myogenic cells modified the action of IL-1β and reduced the ability to proliferate and differentiate. IL-1β induced Jagged1 gene expression may be a feedback response to excess stimulation with this cytokine because high IL-1β (200–1000 pg/ml) induced Jagged1 gene expression even in non-dystrophic myogenic cells. DMD myogenic cells are likely to acquire the susceptibility of the Jagged1 gene to IL-1β under the microcircumstances in DMD muscles. The present results suggest that Jagged1 induced by IL-1β plays a crucial role in the loss of muscle regeneration capacity of DMD muscles. The IL-1β/Jagged1 pathway may be a new therapeutic target to ameliorate exacerbation of muscular dystrophy in a dystrophin-independent manner.</p></div

Human myogenic cells derived from non-dystrophic and dystrophic muscles were immortalized and isolated from non-myogenic cells.

<p>(A-D) Human muscle satellite cells are identified by their M-cadherin⁺/NCAM⁺ status and position under the basal lamina. DAPI, laminin α2, M-cadherin (Mcad), and NCAM were pseudocolored with different colors as indicated at the tops of the panels and then merged. Scale bar, 10 μm. (E and F) NCAM⁺ cells (red squares) were isolated from human immortalized muscle cell cultures using flow cytometry. Hu37KD is immortalized cells derived from a non-dystrophic muscle cell culture (E), whereas DMD1cmv is derived from dystrophic muscle cell culture (F). ALP, alkaline phosphatase. (G and H) Myogenic cells in the NCAM⁺ fraction were isolated. DMD3cmv is immortalized cells derived from a dystrophic muscle cell culture, and then separated into NCAM^- (G) and (H) cells. The cells were cultured for 7 d under the differentiation-inducing condition. Phase contrast images are shown. Arrows represent myotubes. NCAM⁺ cells were designated D3P in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188821#pone.0188821.g004" target="_blank">Fig 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188821#pone.0188821.s006" target="_blank">S2 Table</a>. Scale bar, 50 μm. (I) Procedure of isolation of human myogenic cells from primary cultured muscle cell cultures. (J) Purification and expansion of immortalized human myogenic cells. Primary cultured cells DMD1 derived from dystrophic muscle of a one-year-old boy were immortalized by the three-factor method, and 0.1% of NCAM+ cells were isolated by flow cytometry and expanded in culture. Then, 47% of NCAM+ cells in the primary sorted cell culture were isolated again and expanded. Finally, 96% of secondary sorted cells were NCAM⁺. (K-N) NCAM⁺ cells are desmin-positive myogenic cells. A single cell-derived clone D1P1 was isolated from immortalized dystrophic myogenic cell D1P derived from DMD1 cells. D1P1 expressed desmin (M) and gave rise to prominent myotubes expressing myosin heavy chain (MyHC in N) under the differentiation-inducing condition. Scale bar, 100 μm.</p

IL-1β promoted expression of NF-κB downstream genes in both human dystrophic and non-dystrophic myogenic cells.

<p>(A and B) The expressions of IL-1β (A) and IL-1 receptor (IL1R1) (B) in non-dystrophic (Hu37KD5) and dystrophic (D4P4) myogenic cell clones were analyzed by qRT-PCR. The amounts of mRNA were normalized to control POLR2a mRNA value. The means and standard deviations were estimated from four independent experiments. The statistical significance of the difference between non-dystrophic and dystrophic myogenic cells was analyzed using Student’s <i>t</i>-test. P-values were larger than 0.1 for both genes. (C) Expression profiles of members of the NF-κB signaling pathway in Hu37KD5 (a) and D4P4 (b) were determined using RT-PCR arrays after 24 h of exposure to IL-1β (500 pg/ml). Changes in transcript abundance are expressed as log2 ratio to untreated control mean and designated “Fold regulation”. (D) The expressions of CSF2, CSF3, IL-8, and IL-1β in Hu37KD5 (white column) and D4P4 (black column) were analyzed by qRT-PCR after 24 h exposure to IL-1β (500 pg/ml). The amounts of mRNA were normalized to control POLR2a mRNA values. The means and standard deviations were estimated from at least three independent experiments. Data were statistically analyzed using Student’s <i>t</i>-test. *, p<0.05.</p

Jagged1 and Notch3 genes were upregulated in immortalized human dystrophic myogenic cells.

<p>(A and B) The expression of Notch ligands Jagged1 (JAG1), Jagged2 (JAG2), and deltaless-like1 (DLL1) (A) and Notch receptors Notch1, Notch2, and Notch3 (B) in seven non-dystrophic and six dystrophic myogenic cell lines was analyzed by qRT-PCR. Cells were cultured in growth medium pmGM. The amounts of mRNA were normalized to control B2M mRNA value. The means and standard deviations were estimated from four independent reactions for each cell line. Data were statistically analyzed using Student’s <i>t</i>-test. P values of non-dystrophic myogenic cells and dystrophic myogenic cells are shown at the tops of panels.</p

IL-1β promoted cell cycle progression of non-dystrophic myogenic cells.

<p>(A) Non-dystrophic cells (Hu37KD5) were stimulated with TNFα (500 pg/ml), IL-1β (500 pg/ml), or IL-6 (100 ng/ml) and cultured for six days. The nuclei were counted. The means and standard deviations were estimated from four independent cultures for each treatment. The statistical significance (P-value) of the difference between samples was analyzed using one-way ANOVA. ND, p>0.05; **, p<0.01. (B and C) Non-dystrophic (Hu37KD5, B) and dystrophic (D4P4, C) myogenic cell clones were treated with various concentrations of IL-1β (0–500 pg/ml) for 24 h. The cells were incubated with EdU (10 μM) for further 6 h, and the Click-iT reaction was carried out; then the cells were subjected to fluorescent image analysis. EdU-positive nuclei were counted in nine areas of each culture and the means and standard deviations were estimated. Similar results were obtained from two independent experiments, and representative data are shown. The statistical significance of the difference between the unstimulated control (0 pg/ml) and cytokine-stimulated cells were analyzed using one-way ANOVA. ND, p>0.05; *, p<0.05; **, p<0.01.</p

IL-1β inhibited myogenesis of dystrophic myogenic cells in a Jagged1-dependent manner.

<p>(A-R) Human non-dystrophic (Hu37KD5, A-C) and dystrophic (D4P4, D-F) myogenic cell clones and derivatives of D4P4 were stimulated with (B, C, E, F, H, I, K, and L) or without (A, D, G, and J) IL-1β (500 pg/ml) under the differentiation-inducing condition for 3 d. D4P4 cells were transduced by a lentivirus encoding random shRNA (D4shCTR, G-I) or shJagged1 (D4shJ1, J-L). Myosin heavy chain and troponin T were visualized using an inverted microscope (red). Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (M) Hu37KD5, D4P4, and derivatives of D4P4 were cultured for up to 3 or 4 d under the differentiation-inducing condition with (+) or without (-) IL-1β (500 pg/ml). Twenty micrograms of total proteins was subjected to immunoblot analysis with antibodies against MyHC and β-tubulin.</p

Mechanistic action of IL-1β in human myogenic cells.

<p>(A) A schematic view of the putative action of Jagged1and IL-1β in human myogenic cells. Jagged1 modulates the action of IL-1β in myogenic cells during chronic inflammation in DMD muscles. Then, the Jagged1/Notch3 signaling pathway attenuates the potential of dystrophic myogenic cells to proliferate and differentiate, whereas it is quenched in non-dystrophic myogenic cells. (B) Putative negative feedback effect of Jagged1 in dystrophic myogenic cells. Abundant/chronic exposure to IL-1β, probably derived from macrophages, induces myogenic cells to express IL-1β during repeated inflammation. The autocrine/paracrine IL-1β excessively stimulates myogenic cells, and then induces expression of Jagged1.</p

Extremely high cellular stress upregulated expression of Jagged1 in human non-dystrophic myogenic cells under growing condition.

<p>(A) Non-dystrophic myogenic cells Hu37KDP were cultured in pmGM with or without IL-1β for 24 h. (B) Hu37KDP was cultured in pmGM and exposed to hydrogen peroxide for 30 min and then further cultured in pmGM for 24 h. Twenty micrograms of total proteins was subjected to immunoblot analysis with antibodies against Jagged1. β-tubulin was used as a loading control.</p

Enforced expression of jagged1 interfered with cell cycle progression and antagonized IL-1β.

<p>(A) The non-dystrophic myogenic cell clone Hu37KD5 (white column) was transduced with a control lentivirus vector (Hu37CTR, gray column) or a Jagged1-expression lentivirus (Hu37rJ1, filled column). The amounts of transcripts of Jagged1, Notch1, Notch2, and Notch3 were determined by qRT-PCR. The amounts of mRNA were normalized to control the POLR2a mRNA value. The means and standard deviations were estimated from three independent experiments. (B-C) Twenty micrograms of total proteins from Hu37CTR (CTR) and Hu37rJ1 (J1) were subjected to immunoblotting with antibodies against Jagged1 (B) and Notch3 (C). F, Unprocessed full-length protein; CTF, cytoplasmic fragment; asterisks, non-specific bands. Numbers represent the positions of protein size markers. (D) Hu37CTR (white circles) and Hu37rJ1 (filed circles) cell clones were treated with IL-1β (0–250 pg/ml) for 24 h. The cells were incubated with EdU (10 μM) for a further 6 h culture, and then subjected to fluorescence analysis. EdU-positive nuclei were counted in nine areas of each culture and the means and standard deviation were estimated. Data were statistically analyzed using one-way ANOVA. The statistical significance of the difference between the unstimulated control (0 pg/ml) and cytokine-stimulated cells (60–250 pg/ml) was less than 0.01 in both Hu37CTR and Hu37rJ1. The statistical significance of the difference between Hu37CTR and Hu37rJ1 was less than 0.01 at each dosage of IL-1β. (E) The expression of CSF2 in Hu37CTR (white column) and Hu37rJ1 (gray column) cells was analyzed by qRT-PCR after 24 h of exposure to IL-1β. The amounts of mRNA were normalized to the control POLR2a mRNA value. The means and standard deviations were estimated from three independent experiments. Data were statistically analyzed using Student’s <i>t</i>-test. *, p<0.05.</p