Stress-induced cell death in control and LSFC fibroblasts.

<p><b>(A)</b> Lactate dehydrogenase (LDH) release in control and LSFC fibroblasts exposed for 48 h to factors relevant to acidotic crises. <b>(B)</b> Mean LDH release (<i>n</i> = 5), <b>(C)</b> Caspase 3/7 activity (<i>n</i> = 7), <b>(D)</b> Cellular ATP content (<i>n</i> = 7) and <b>(E)</b> COX/CS activity ratio (<i>n</i> = 3) assessed at baseline and after exposure to PL (palmitate 1 mM; lactate 10 mM) for 34 h (LDH release) or 24 h (other parameters). All experiments were performed in one control (EBS-4) and one LSFC (AL-006) cell line. Data are expressed as means ± S.E. Statistical significance of differences between groups or conditions was assessed with a two-way ANOVA for repeated measures followed by a Bonferroni post hoc test. Significantly different from the control cells in the same experimental condition: * <i>p</i> < 0.05, ** <i>p</i> < 0.01. Significantly different from baseline within the same experimental group: ^ρ<i>p</i> < 0.05, ^ρρ<i>p</i> < 0.01, ^ρρρ<i>p</i> < 0.001.</p

Effect of the LRPPRC A354V mutation on basal mitochondrial network morphology and functions.

<p>Representative live cell images of MTG-loaded control <b>(A)</b> and LSFC <b>(B)</b> fibroblasts used for quantitative analysis of mitochondrial network morphology. <b>(C)</b> Form Factor (FF) values calculated using the equation FF = 4π*Area/perimeter2 (n = 6). Representative live cell images of control <b>(D)</b> and LSFC <b>(E)</b> fibroblasts labeled with TMRE (red) and MTG (green). <b>(F)</b> Mitochondrial membrane (ΔΨ) potential expressed as the ratio of TMRE to MTG (n = 5). Lower values are indicative of reduced ΔΨ. <b>(G)</b> Mean fluorescence intensity of the mitochondria-specific superoxide probe MitoSOX in control and LSFC fibroblasts (n = 5). <b>(H)</b> Maximal ADP-driven respiration in digitonin-permeabilized fibroblasts energized with complex I (5 mM glutamate—2.5 mM malate; Glut-Mal; n = 15) or complex II substrates in presence of the complex I inhibitor rotenone (5 mM succinate + 1 μM rotenone; Succ+Rot; n = 14). Inset shows representative respirometry traces confirming that respiratory rates increased promptly in response to the addition of respiratory substrates, and were potently inhibited by complex I (rotenone), and complex II (malonate) blockers. <b>(I)</b> Mitochondrial calcium retention capacity (CRC) in control and LSFC fibroblasts exposed to progressive Ca2+ loading (n = 8). Inset shows representative Ca2+ kinetic tracings observed in control and LSFC fibroblasts. Tracings show progressive Ca2+ accumulation followed by PTP-induced release of accumulated Ca2+. Each spike indicates the addition of a calcium pulse of 83 nmoles. All experiments were performed in one control (EBS-4) and one LSFC (AL-006) cell line, except for the determination of ΔΨ, which was performed in EBS-3 and AL-002. Data are expressed as means ± S.E. Difference between control and LSFC cells was assessed with a paired t-test. Significantly different from the control group: * <i>p</i> < 0.05, ** <i>p</i> ≤ 0.01. Statistical power: C: 92%; F: 85%; G: 80%; H: Glut-Mal 80%; Succ+Rot: 96%; I: 73%.</p

Control and Leigh Syndrome French Canadian (LSFC) skin fibroblast cell lines.

<p>Control and Leigh Syndrome French Canadian (LSFC) skin fibroblast cell lines.</p

Effect of the LRPPRC A354V mutation on LRPPRC content, mitochondrial content and cellular ATP levels.

<p><b>(A)</b> Immunoblot and densitometric analysis of LRPPRC content in whole cell lysates from control and LSFC fibroblasts (n = 4). Representative immunofluorescence images of control <b>(B;C)</b> and LSFC fibroblasts <b>(D;E)</b> labeled with anti-LRPPRC (green) and anti-pyruvate dehydrogenase (red) antibodies. Overlay images (yellow), and line scan analysis show the cellular distribution of LRPPRC in the mitochondrial compartment. <b>(F)</b> Cytochrome c oxidase (COX) enzyme activity in whole cell lysates from control and LSFC fibroblasts was normalized to that of the mitochondrial marker citrate synthase (CS) to take into account possible differences in mitochondrial content (n = 4). <b>(G)</b> Immunoblot and densitometric analysis of VDAC, a mitochondrial marker protein, in whole cell lysates from control and LSFC fibroblasts (n = 3). <b>(H)</b> Citrate synthase activity in whole cell lysates from control and LSFC fibroblasts (n = 4). <b>(I)</b> Cellular ATP content in control and LSFC fibroblasts (n = 4). Data are expressed as means ± S.E. Experiments were performed in one control (EBS-4) and one LSFC cell line (AL-006). Difference between control and LSFC cells was assessed with a paired t-test. ** Significantly different from the control group <i>p</i> ≤ 0.01. Statistical power for LRPPRC and COX was 97%.</p