mTORC1 is required for expression of LRPPRC and cytochrome- c oxidase but not HIF-1α in Leigh syndrome French Canadian type patient fibroblasts

Abstract

Leigh syndrome French Canadian type (LSFC) is a mitochondrial disease caused by mutations in the leucine-rich pentatricopeptide repeat-containing (LRPPRC) gene leading to a reduction of cytochrome-c oxidase (COX) expression reaching 50% in skin fibroblasts. We have shown that under basal conditions, LSFC and control cells display similar ATP levels. We hypothesized that this occurs through upregulation of mechanistic target of rapamycin (mTOR)-mediated metabolic reprogramming. Our results showed that compared with controls, LSFC cells exhibited an upregulation of the mTOR complex 1 (mTORC1)/p70 ribosomal S6 kinase pathway and higher levels of hypoxia-inducible factor 1α (HIF-1α) and its downstream target pyruvate dehydrogenase kinase 1 (PDHK1), a regulator of mitochondrial pyruvate dehydrogenase 1 (PDH1). Consistent with these signaling alterations, LSFC cells displayed a 40-61% increase in [U-¹³C₆]glucose contribution to pyruvate, lactate, and alanine formation, as well as higher levels of the phosphorylated and inactive form of PDH1-α. Interestingly, inhibition of mTOR with rapamycin did not alter HIF-1α or PDHK1 protein levels in LSFC fibroblasts. However, this treatment increased PDH1-α phosphorylation in control and LSFC cells and reduced ATP levels in control cells. Rapamycin also decreased LRPPRC expression by 41 and 11% in LSFC and control cells, respectively, and selectively reduced COX subunit IV expression in LSFC fibroblasts. Taken together, our data demonstrate the importance of mTORC1, independent of the HIF-1α/PDHK1 axis, in maintaining LRPPRC and COX expression in LSFC cells.

Keywords: COX; LRPPRC; LSFC; mTORC1; mitochondrial diseases; rapamycin.

Fig. 1.

Higher contribution of exogenous glucose to pyruvate formation in Leigh syndrome French Canadian type (LSFC) fibroblasts. A and B: immunoblot and densitometric analysis of leucine-rich pentatricopeptide repeat-containing (LRPPRC, n = 12; A) and cytochrome-c oxidase subunit IV (COX IV, n = 9; B) protein levels in primary fibroblasts from LSFC and control donors. C and D: immortalized control and LSFC fibroblasts were incubated with 5 mM [U-¹³C₆]glucose, 0.2 mM pyruvate, and 0.5 mM glutamine for 4 h in serum-free DMEM, and the relative contribution of glucose to the formation of pyruvate, lactate, and alanine was analyzed by GC-MS (n = 7). Results represent means ± SE. Difference between control and LSFC cells was assessed with a paired Student’s t-test. *P ≤ 0.05; ***P ≤ 0.001.

Fig. 2.

Upregulation of the Akt/mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway in Leigh syndrome French Canadian type (LSFC) fibroblasts. A: AMPK activity of primary fibroblasts from control and LSFC donors was assessed by immunoblot and densitometric analysis of total and phosphorylated levels of AMPK (n = 7) and its downstream target acetyl-CoA carboxylase (ACC; n = 6). B and C: activity of the mTORC1 signaling pathway in control and LSFC primary fibroblasts was assessed by immunoblot and densitometric analysis of total and phosphorylated levels of Akt (n = 10), mTOR (n = 13), and their downstream targets p70 ribosomal S6 kinase (p70S6K, n = 9) and eukaryotic translation initiation factor 4E-binding protein-1 (4E-BP1, n = 12). Results represent means ± SE. Difference between control and LSFC cells was assessed with a paired Student’s t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 3.

Pyruvate dehydrogenase 1 (PDH1) is inhibited in Leigh syndrome French Canadian type (LSFC) fibroblasts. Immunoblot and densitometric analysis of total and/or phosphorylated levels of hypoxia-inducible factor 1α (HIF-1α, n = 5) and pyruvate dehydrogenase kinase 1 (PDHK1, n = 5; A) and PDH1-α (n = 8; B) in primary fibroblasts from control and LSFC donors. Results represent means ± SE. Difference between control and LSFC cells was assessed with a paired Student’s t-test. *P ≤ 0.05.

Fig. 4.

Suppression of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) activity does not alter hypoxia-inducible factor 1α (HIF-1α) and pyruvate dehydrogenase kinase 1 (PDHK1) levels. Primary fibroblasts from control and Leigh syndrome French Canadian type (LSFC) donors were treated for 16 h with 100 nM rapamycin or vehicle (DMSO) in supplemented DMEM. On the day of the study, cells were cultured in a serum-free nonsupplemented DMEM, with inhibitor or vehicle as indicated, for an additional 4 h. A: suppression of mTORC1 activity was confirmed by immunoblot and densitometric analysis of phosphorylated levels of mTOR (n = 3), p70 ribosomal S6 kinase (p70S6K, n = 3), and Akt (n = 3). B and C: immunoblot and densitometric analysis of total levels of HIF-1α (n = 7) and PDHK1 (n = 8; B) and phosphorylated pyruvate dehydrogenase 1α (PDH1-α, n = 7; C) in response to rapamycin treatment. D: primary fibroblasts were cultured with inhibitor or vehicle, as indicated, in opaque 96-well plates at a density of 10,000 cells per well in triplicate. Cellular ATP levels were measured with the ATPlite luminescence ATP detection assay system (n = 4). Results represent means ± SE. Difference between baseline and rapamycin treatment was assessed with a paired Student’s t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 5.

Suppression of mechanistic target of rapamycin complex 1 (mTORC1) activity decreases leucine-rich pentatricopeptide repeat-containing (LRPPRC) and cytochrome-c oxidase (COX) expression in Leigh syndrome French Canadian type (LSFC) fibroblasts. Primary fibroblasts from control and LSFC donors were treated for 16 h with 100 nM rapamycin or vehicle (DMSO) in supplemented DMEM. On the day of the study, cells were cultured in serum-free nonsupplemented DMEM, with inhibitor or vehicle as indicated for an additional 4 h. A: immunoblot and densitometric analysis of mitochondrial complex levels [NADH dehydrogenase (ubiquinone) iron-sulfur protein-2, mitochondrial (NDUFS2, n = 5), succinate dehydrogenase (ubiquinone) iron-sulfur subunit, mitochondrial (SDHB, n = 3), cytochrome–b-c1 complex subunit 2, mitochondrial (UQCRC2, n = 4), and ATP synthase subunit-α, mitochondrial (ATP5A, n = 5)] in response to rapamycin treatment. B: immunoblot and densitometric analysis of LRPPRC (n = 8) and COX subunit IV (COX IV, n = 11) content in response to rapamycin treatment. Results represent means ± SE. CI–CV, oxidative phosphorylation complexes I–V. Difference between baseline and rapamycin treatment was assessed with a paired Student’s t-test. *P ≤ 0.05; **P ≤ 0.01.