Fig 1: mtDNA accumulation is responsible for inflammation and rhabdomyolysis that can be reversed by steroids or chloroquine (CLQ) treatment in vivo(A) Real-time calcium flux in myoblasts from 4 controls and patients challenged with CpG-A, after pre-incubation with ODN151, a synthetic TLR9 antagonist. Each dot plot with lines depicts the mean ± SD of the intensity of the fluo-4 calcium probe as normalized to basal intensity of 1 single individual (=10 cells/condition).(B) Death of myoblasts was evaluated by staining cells from 4 control individuals transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein (+LPIN1) and 4 patients transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein or an empty vector, for the apoptosis marker caspase3/7 green and propidium iodide or 7-actinomycin D (7-AAD), a dye that stains both apoptotic and necrotic cells. Staurosporin (ST): positive control. Fluorescence-activated cell sorting (FACS) plots show the codistribution of the fluorescence intensity of caspase3/7 and of 7-AAD in 1 of 4 controls and patients.(C) Dot plots (=1,000 single cells/dot) depict the mean ± SD of the proportion of dead cells (ie, cells positive for capsase3/7 and/or 7-AAD) from (B) (mean effect of interaction F(8,45) = 5.296, p = 0.0001, of stimulus F(4,45) = 70.03, p < 0.0001, of subjects F(2,45) = 6.607, p = 0.003).(D) As in (C), but using immortalized myoblast cell line transduced with Sh-Sc or Sh-LPIN1 shRNA (mean effect of interaction F(4,20) = 6.154, p = 0.0021, of stimulus F(4,20) = 48.93, p < 0.0001, of subjects F(1,20) = 15.62, p = 0.0008).(E and F) As in (C), but myoblasts were pre-treated with Et. B. or vehicle (mean effect of interaction F(3,24) = 21.28, p < 0.0001, of stimulus F(3,24) = 114.8, p < 0.0001, of subjects F(1,24) = 25.79, p < 0.0001) for 5 days (E) or dexamethasone (DXM) or vehicle (mean effect of interaction F(3,24) = 9.490, p = 0.0003, of stimulus F(3,24) = 33.95, p < 0.0001, of subjects F(1,24) = 11.56, p = 0.0024) for 8 h (F), before challenging myoblasts with CpG-A in the presence of GM or EBSS.(G) CK levels (means ± SDs of 3 individual values obtained from independent blood samples drawn the same day) were measured in 2 patients during a flare before and after the administration of steroids.(H) As in (C) and Figure 5, but myoblasts from 4 controls and patients were pre-treated with CLQ or ODN151, or vehicle for 8 h followed by challenging with CpG-A together with GM or EBSS for another 16 h (mean effect of interaction F(5,36) = 19.53, p < 0.0001, of stimulus F(5,36) = 38.32, p < 0.0001, of subjects F(1,36) = 67.60, p < 0.0001). Dot plots (mean of 4–6 technical replicates/dot) show the mean ± SD of IL-6 concentration at the end of the experiment.(I) As in (H), but evaluating the proportion of cell death (mean effect of interaction F(5,36) = 17.51, p < 0.0001, of stimulus F(5,36) = 73.41, p < 0.0001, of subjects F(1,36) = 40.06, p < 0.0001). Dot plots (=1,000 single cells/dot) show the means ± SDs. IL-6 concentration is presented after background (which corresponds to values obtained from culture supernatants of cells exposed to vehicle) subtraction.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001: adjusted p values as determined by a between-subjects 2-way ANOVA (C–F, H, and I) and post hoc Sidak’s correction for multiple comparisons. Results are from 1 representative of 2 (D) or at least 3 (A–C, E–I) independent experiments.
Fig 2: Lipin1 localizes in proximity of late endosomes, autophagosomes, and mitochondria in human myoblasts(A) The confocal images show a representative staining of endogenous lipin1 in primary myoblasts from 1 of 6 controls and 6 patients carrying LPIN1 mutations (Table S1) and in immortalized myoblasts from 1 healthy donor transduced with sh-LPIN1 or control scrambled (sh-Sc) shRNA.(B) Immunoblot analysis of endogenous lipin1 expression as in (A) in 5 patients.(C) Dot plots (mean of 3 technical replicates/dot) depict the means ± SDs of PAP activity quantified in myoblasts from 6 controls and 5 patients (Mann-Whitney U test).(D) Lipin1 distribution within myoblast organelles of 5 controls by confocal microscopy. Dot plots (50–80 cells/dot) show the means ± SDs of the percentage of lipin1 in proximity with a given protein. The images are representative of 1 of at least 3 independent experiments.Scale bars, 10 µm.
Fig 3: Autophagic clearance and mitochondrial quality and functions are impaired in human lipin1-deficient myoblasts(A) Autolysosome formation (red puncta) in myoblasts transfected with the RFP-GFP-LC3 construct upon exposure to EBSS and refed or not with GM. Box and whisker plots (50 images/condition) represent the percentage of autolysosomes (mean effect of interaction F(1,196) = 44.4, p < 0.0001, of stimulus F(1,196) = 47.63, p < 0.0001, of subjects F(1,196) = 17.78, p < 0.0001).(B) Myoblasts were cultured in GM or EBSS and refed or not with GM (starvation-induced mitophagy) or exposed to CCCP before immunostaining for TOMM20 and LC3. Box and whisker plots (50 images/condition) depict the percentage of proximity of TOMM20 with LC3 (mean effect of interaction F(3,392) = 11.09, p < 0.0001, of stimulus F(3,392) = 62.9, p < 0.0001, of subjects F(1,392) = 20.70, p < 0.0001).(C) Box and whisker plots (30 images/condition) represent the number of mitochondrial nucleoids per myoblast after exposure to CCCP (mean effect of interaction F(3,116) = 9.554, p < 0.0001, of time F(3,116) = 19.99, p < 0.0001, of subjects F(1;116) = 26.22, p < 0.0001).(D) Real-time analysis of oxygen consumption rate (OCR) in myoblasts under basal respiration and after addition of (1) oligomycin, (2) FCCP, or (3) antimycin A. Dot plot (mean of 4–6 technical replicates/dot) represents the means ± SDs of the OCR in myoblasts from 5 controls and 4 patients.(E) Evaluation of the mitochondrial membrane potential by flow cytometry. Dot plot (mean of 2 technical replicates/dot) depicts the means ± SDs of the MFI of TMRM, expressed as a percentage of the MFI for GM condition (mean effect of interaction F(1,12) = 3.340, p = 0.0926, of stimulus F(1,12) = 7.313, p = 0.0192, of subjects F(1,12) = 0.2404, p = 0.6327).(F) Distribution of oxidized DNA within the LC3 and LAMP1 structures of myoblasts cultured as in (B). Box and whisker plots (25 images/condition) show the percentage of proximity of 8OHDG with LC3 (mean effect of interaction F(2,144) = 1.504, p = 0.2258, of stimulus F(2,144) = 0.2997, p = 0.7415, of subjects F(1,144) = 100.8, p < 0.0001) or LAMP1 (mean effect interaction F(2,144) = 3.130, p = 0.0467, of stimulus F(2,144) = 18.41, p < 0.0001, of subjects F(1,144) = 96.93, p < 0.0001).(G) Quantification of 12S mtDNA levels qPCR in cytosolic fractions from myoblasts of 4 patients and controls exposed to CCCP or EBSS (mean effect of interaction F(1,12) = 0.004867, p = 0.9455, of stimulus F(1,12) = 1.798, p = 0.2047, of subjects F(1,12) = 0.03755, p = 0.8496) and of the mtDNA motif DLOOP (mean effect of interaction F(1,12) = 0.2168, p = 0.6498, of stimulus F(1,12) = 0.3160, p = 0.5844, of subjects F(1,12) = 3.371, p = 0.0913). Dot plots (mean of 3 technical replicates/dot) depict the means ± SDs of the ratio of cycle threshold (CT) values of the given cytosolic fraction normalized to unfractioned cells.Scale bars, 10 µm (A and B). *p < 0.05, **p < 0.01, ***p < 0.001: adjusted p values after between-subjects (A, B, and E–G) or within-subjects (C, as compared to H0 for each family) 2-way ANOVA and post hoc Sidak’s correction for multiple comparisons. Images and plots show typical staining and quantification for 1 of 3 patients and controls (A, B, C, and F). Results are representative from 1 of 2 (G), 3 (A–D and F), and 4 (E) independent experiments.See also Figures S3 and S4.
Fig 4: Lipin1 deficiency results in a loss of PI3P close to Rab7 structures in myoblasts(A) The confocal images show a representative 2xFYVE-GFP staining from 1 of 6 controls and patients. Dot plots (50 images/dot) show the means ± SDs number of 2xFYVE-GFP dots per patient versus control myoblast (Mann-Whitney U test).(B) As in (A), but using immortalized myoblasts from 1 healthy donor treated with sh-Sc or sh-LPIN1. The plots (30–45 cells/condition) show the number of 2xFYVE-GFP dots (Mann-Whitney U test).(C) Typical distribution of PI3P within the myoblast vesicles from 1 of 3 controls and patients. Box and whisker plots (50 images/condition) depict the percentage of proximity between PI3P and the given marker for 1 representative control and patient (Mann-Whitney U test).(D) Myoblasts from 3 controls and 3 patients were stained for PI35P2 and late endosomal or lysosomal markers. Proximity was calculated as in (C) (unpaired t test). Results are from 1 representative of at least 3 independent experiments.Scale bars, 10 µm.See also Figure S1.
Fig 5: Accumulation of oxidized mitochondria enhances inflammation in lipin1-deficient cells through a TLR9 pathway(A) Circulating mtDNA was quantified in plasma by qPCR and inflammatory molecules were measured in sera by flow cytometry, from 12 healthy donors (6 for mtDNA) and 6 patients (Mann-Whitney U test). Dot plots (mean of 3 technical replicates/dot) show the means ± SDs.(B) IFN signature in peripheral blood mononuclear cells was identified by qPCR in 4 controls and 4 patients. Dot plots (mean of 3 technical replicates/dot) show the mean ± SD of the CT value of a given gene normalized to the CT value of BACT (Mann-Whitney U test).(C) DC maturation reflected by CD83 expression (mean effect of interaction F(4,70) = 2.114, p = 0.0881, of stimulus F(4,70) = 36.27, p < 0.0001, of subjects F(1,70) = 14.21, p = 0.0003) and interleukin-6 (IL-6) production (mean effect of interaction F(3,64) = 2.847, p = 0.0444, of stimulus F(3,64) = 18.20, p < 0.0001, of subjects F(1,64) = 2.807, p = 0.0987) were evaluated by flow cytometry and ELISA, respectively, in 10 controls and 6 patients, after exposure to Poly:IC (PIC), lipopolysaccharide (LPS), imiquimod (Im), and CpG-A. Dot plots (mean of 2 [CD83] or 4 [IL-6] technical replicates/dot) show the means ± SDs.(D) CpG-A-induced IFN responses in DCs from 3 controls and patients were evaluated by qPCR by quantifying SIGLEC1 (mean effect of interaction F(1,8) = 4.436, p = 0.0683, of stimulus F(1,8) = 9.255, p = 0.0160, of subjects F(1,8) = 20.78, p = 0.0019), ISG15 (mean effect of interaction F(1,8) = 0.3191, p = 0.5876, of stimulus F(1,8) = 21.75, p = 0.0016, of subjects F(1,8) = 41.33, p = 0.0002), and IFI27 (mean effect of interaction F(1,8) = 21.99, p = 0.0016, of stimulus F(1,8) = 25.45, p = 0.0010, of subjects F(1,8) = 91.20, p < 0.0001) expression. Dot plots (mean of 3 technical replicates/dot) show the means ± SDs of CT value calculated as in (B).(E) Production of IL-6 (ELISA) by myoblasts from 8 controls and 6 patients (mean effect of interaction F(2,36) = 3.882, p = 0.0297, of stimulus F(2,36) = 45.49, p < 0.0001, of subjects F(1,36) = 15.28, p = 0.0004), IFNA (mean effect of interaction F(3,24) = 23.44, p < 0.0001, of stimulus F(3,24) = 74.13, p < 0.0001, of subjects F(1,24) = 42.33, p < 0.0001), and IFNB (mean effect of interaction F(3,24) = 2.207, p = 0.1133, of stimulus F(3,24) = 10.19, p = 0.0002, of subjects F(1,24) = 13.60, p = 0.0012).(F) As in (E), but using sh-Sc or sh-LPIN1-transduced immortalized myoblasts from 1 healthy donor (1 different cell vial from the same single healthy donor/dot) and primary myoblasts from 4 patients. Dot plots (mean of 4 technical replicates/dot) show the means ± SDs of the IL-6 concentration (mean effect of interaction F(2,14) = 6.234, p = 0.0116, of stimulus F(1,14) = 0.0874, p = 0.3653, of subjects F(2,14) = 4.397, p = 0.0330).(G) As in (F), but primary myoblasts from 4 controls and patients were transduced with a lentivirus expressing a plasmid encoding for a myc-DDK-tagged lipin1 protein (+LPIN1) or an empty vector (+vector) before being challenged (mean effect of interaction F(2,18) = 0.2196, p = 0.8050, of stimulus F(1,18) = 0.2906, p = 0.5965, of subjects F(2,18) = 14.80, p = 0.0002).(H) Myoblasts from 6 controls and patients were exposed to a vehicle or ethidium bromide (Et. B.). Dot plots (mean of 3 technical replicates/dot) show the means ± SDs of the ratio of the CT value for 12S mitochondrial DNA normalized to the CT value for BACT (mean effect of interaction F(1,12) = 3.186, p = 0.0996, of stimulus F(1,12) = 101.6, p < 0.0001, of subjects F(1,12) = 5.268, p = 0.0405).(I) Myoblasts from 6 controls and patients pre-treated with Et. B. for 5 days were exposed to EBSS and challenged or not with CpG-A for 16 h. Dot plots (mean of 4 technical replicates/dot) show the mean ± SD of IL-6 concentration in culture supernatants at the end of the experiment (mean effect of interaction F(3,24) = 4.989, p = 0.0079, of stimulus F(3,24) = 8.218, p = 0.0006, of subjects F(1,24) = 15.14, p = 0.0007).(J) Myoblasts were transfected with a plasmid encoding HA-tagged TLR9 and exposed to GM or EBSS and with vehicle or CpG-A before refeeding (Refed) cells exposed to EBSS and CpG-A with GM. Cells were then stained for HA-TLR9 (anti-HA antibody, red) and 8OHDG (green). Box whisker plots (25 images/condition) show the percentage of proximity of TLR9 with 8OHDG (mean effect of interaction F(4,240) = 1.712, p = 0.1481, of stimulus F(4,240) = 25.07, p < 0.0001, of subjects F(1,240) = 68.55, p < 0.0001) in myoblasts from 1 of 3 controls and patients.(K) As in (J) but myoblasts were immunostained for HA-TLR9 (green) and LC3 (red). Mean effect of interaction F(4,240) = 18.56, p < 0.0001, of stimulus F(4,240) = 125.8, p < 0.0001, of subjects F(1,240) = 61.39, p < 0.0001.IL-6 concentration is presented after background (which corresponds to values obtained with a control vehicle) subtraction (C, E, F, H, and I). Scale bars, 10 µm (J and K). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001: adjusted p values after between-subjects 2-way ANOVA (B–K) and post hoc Sidak’s correction for multiple comparisons. Data are representative of 1 of at least 3 independent experiments.See also Figure S5.
Supplier Page from OriGene Technologies for Lipin 1 (LPIN1) Human shRNA Lentiviral Particle (Locus ID 23175)