Deletion of the cardiolipin-specific phospholipase Cld1 rescues growth and life span defects in the tafazzin mutant: implications for Barth syndrome

Cardiolipin (CL) that is synthesized de novo is deacylated to monolysocardiolipin (MLCL), which is reacylated by tafazzin. Remodeled CL contains mostly unsaturated fatty acids. In eukaryotes, loss of tafazzin leads to growth and respiration defects, and in humans, this results in the life-threatening disorder Barth syndrome. Tafazzin deficiency causes a decrease in the CL/MLCL ratio and decreased unsaturated CL species. Which of these biochemical outcomes contributes to the physiological defects is not known. Yeast cells have a single CL-specific phospholipase, Cld1, that can be exploited to distinguish between these outcomes. The cld1Δ mutant has decreased unsaturated CL, but the CL/MLCL ratio is similar to that of wild type cells. We show that cld1Δ rescues growth, life span, and respiratory defects of the taz1Δ mutant. This suggests that defective growth and respiration in tafazzin-deficient cells are caused by the decreased CL/MLCL ratio and not by a deficiency in unsaturated CL. CLD1 expression is increased during respiratory growth and regulated by the heme activator protein transcriptional activation complex. Overexpression of CLD1 leads to decreased mitochondrial respiration and growth and instability of mitochondrial DNA. However, ATP concentrations are maintained by increasing glycolysis. We conclude that transcriptional regulation of Cld1-mediated deacylation of CL influences energy metabolism by modulating the relative contribution of glycolysis and respiratio

Effects on cell viability and proliferation.

<p>(A) Phase contrast image of neurospheres at passage 3 from the cerebral cortex of E14.5 C57BL/6 mice. (B) Immunofluorescence images of neurospheres stained for nestin and DAPI. (C) Quantitative analysis of the viability of NSCs exposed to the harvesting media or the PCM for 0, 1, 2, 4, 6, 8, 12, 16 or 24 h. (D) Quantitative analysis of S-phase entry (percentage of BrdU-positive cells) in dissociated NSCs exposed to the harvesting media or the PCM for 0, 1, 2, 4, 6, 8, 12, 16 or 24 h. (E) Images of cell cycle analysis by flow cytometry of NSCs exposed to the harvesting media or the PCM for 6 h. (F) Quantitative analysis of S-phase entry (percentage of BrdU-positive cells) in dissociated NSCs exposed to the harvesting media for 6 h and successively cultured in the PCM for another 24 h. (G) Quantitative analysis of S-phase entry (percentage of BrdU-positive cells) in dissociated NSCs exposed to the harvesting media for 8 h and successively cultured in the PCM for another 24 h. (H) Quantitative analysis of S-phase entry (percentage of BrdU-positive cells) in dissociated NSCs exposed to the harvesting media for 16 h and successively cultured in the PCM for another 24 h. For (C) and (D), data are presented as mean ±SEM; n = 5 per group per time point. For (F), (H) and (G), data are presented as mean ±SEM; ns, nonsignificant; ***, p<0.001; n = 5 per group. Scale bar: (A)  = 100 µm; (B)  = 50 µm. Abbreviation: BrdU, 5-bromo-2-deoxyuridine; DAPI, 4′,6′-diamidino-2-phenylindole; E14.5, embryonic day 14.5; NSC, neural stem cell; PCM, proliferation culture medium; SEM, standard error of the mean.</p

Effects on apoptosis and necrosis of NSCs.

<p>(A) Images of flow cytometry analysis of Annexin-V and PI staining of NSCs exposed to the harvesting media or the PCM for 12 h. (B) Images of TUNEL staining of NSCs exposed to the harvesting media or the PCM for 16 h. (C–D) Quantitative analysis of the percentage of apoptotic (C) and necrotic cells (D) in NSCs exposed to the harvesting media or the PCM for 12 h. Data are presented as mean ±SEM. ***, p<0.001 versus PCM. n = 5 per group. (E) Quantitative analysis of TUNEL-positive NSCs exposed to the harvesting media or the PCM for 16 h. Data are presented as mean ±SEM. ns, nonsignificant; ***, p<0.001 versus PCM. n = 10 per group. (F) TEM images of NSCs exposed to the harvesting media or the PCM for 12 h. For Saline, arrow denotes a loss of cytoplasm and evident karyopyknosis and nuclear fragmentation; for PBS, arrow denotes a loss of cytoplasm and evident karyopyknosis; for ACSF, arrow denotes apoptotic bodies and a loss of cytoplasm; for PCM, arrow denotes an intact nuclear envelope and uniformly dispersed chromatin. Scale bar: (C)  = 100 µm; (F)  = 1 µm. Abbreviation: NSC, neural stem cell; PCM, proliferation culture medium; PI, propidium iodide; SEM, standard error of the mean; TEM, transmission electron microscopy; TUNEL, terminal deoxynucleotidyl transferase (TdT) dilutions of cell-delivered dUTP nick 3′-end DNA labeling.</p

Mechanism of harvesting media exposure-induced proliferation inhibition.

<p>(A) Western blots of p53 and cyclin E1 from NSCs exposed to the harvesting media or the PCM for 6 h. β-actin was used as a loading control. (B) Images of BrdU incorporation assays of NSC-neurospheres exposed to the harvesting media or the PCM for 1 week. (C–D) Quantitative analysis of the relative levels of p53 (C) and cyclin E1 (D) to β-actin in NSCs exposed to the harvesting media or the PCM for 6 h. Data are presented as mean ±SD. n = 5 per group. bdi, below detectable limit; *, p<0.05; **, p<0.01; ***, p<0.001 versus PCM. (E) Quantitative analysis of S-phase entry (percentage of BrdU-positive cells) of NSCs in neurospheres exposed to the harvesting media or the PCM for 1 week. The box plots have Min to Max whiskers, a line for the median, and edges for the minimum and maximum. ns, nonsignificant; n = 5 per group. Scale bar: (B)  = 100 µm. Abbreviation: bdi, below detectable limit;BrdU, 5-bromo-2-deoxyuridine; NSC, neural stem cell; PCM, proliferation culture media; SD, standard deviation; SEM, standard error of the mean.</p

Effects on the synaptogenesis by graft-derived cells.

<p>(A) Immunofluorescence images of TBI murine cerebral cortex receiving the harvesting media or PCM-exposed NSC grafting for 21 days and stained for GFP and synaptophysin. (B) Quantitative analysis of percentage of synaptophysin-positive cells in GFP-positive graft-derived cells.. Data are presented as mean ±SEM; *, p<0.05; ***, p<0.001. (C) Quantitative analysis of fluorescence intensity of graft-derived synaptophysin. Data are presented as mean ±SD; ***, p<0.001. Scale bar: (A)  = 25 µm. Abbreviation: GFP, green fluorescence protein; NSC, neural stem cell; PCM, proliferation culture medium; SD, standard deviation; SEM, standard error of deviation; TBI, traumatic brain injury.</p

MMP analysis of harvesting media-exposed NSCs.

<p>(A) Confocal images of dissociated NSCs exposed to the harvesting media, the PCM or 5 µM staurosporine (positive control) for 12 h and stained for MMP. (B) Quantitative analysis of average fluorescence intensity of MMP of NSCs exposed to the harvesting media, the PCM or 5 µM staurosporine (positive control) for 12 h. Data are presented as mean ±SD; #, p<0.001 versus STS; ns, nonsignificant versus STS; n = 5 per group. No significant difference was detected between the harvesting media-exposed NSCs and PCM-exposed NSCs. Scale bar: (A)  = 100 µm. Abbreviation: ACSF, artificial cerebrospinal fluid; MMP, mitochondrial membrane potential; NSC, neural stem cell; PCM, proliferation culture medium; SD, standard deviation; STS, staurosporine.</p

Western blot analysis of apoptosis-related molecules in harvesting media-exposed NSCs.

<p>(A) Western blots of Fas-L, cleaved caspase 8 and 3 from NSCs exposed to the harvesting media or the PCM for 12 h. β-actin was used as a loading control. (B) Western blots of cleaved caspase 9 from NSCs exposed to the harvesting media, the PCM or 5 µM staurosporine (positive control) for 12 h. β-actin was used as a loading control. (C–D) Quantitative analysis of the levels of Fas-L (C), cleaved caspase 3 (D) and cleaved caspase 8 (E) relative to β-actin in NSCs exposed to the harvesting media or the PCM for 12 h. Data are presented as mean ±SD. n = 5 per group. bdi, below detectable limit; *, p<0.05; **, p<0.01; ***, p<0.001 versus PCM. Abbreviation: ACSF, artificial cerebrospinal fluid; bdi, below detectable limit; NSC, neural stem cell; PCM, proliferation culture medium; SD, standard deviation; STS, staurosporine.</p

Effects on motor function recovery.

<p>(A) Quantitative analysis of the maximal speeds of mice in the rotarod test. (B) Quantitative analysis of foot faults per 50 steps of mice when walking across the beam. For (A) and (B), data are presented as mean ±SD; #, p<0.001 versus PCM-exposed NSC. n = 6 per group per time point. Transplantation was conducted 7 days post-TBI. Abbreviation: SD, standard deviation; TBI, traumatic brain injury.</p