Limits...
Mitochondrial vulnerability and increased susceptibility to nutrient-induced cytotoxicity in fibroblasts from leigh syndrome French canadian patients.

Burelle Y, Bemeur C, Rivard ME, Thompson Legault J, Boucher G, LSFC ConsortiumMorin C, Coderre L, Des Rosiers C - PLoS ONE (2015)

Bottom Line: This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production.Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol), or modulate fatty acid (L-carnitine) or Krebs cycle metabolism (propionate) are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol) exacerbate palmitate plus lactate-induced cell death.Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise questions about the nature of the diets, particularly excess fat intake, as well as on the use of antioxidants in patients with LSFC and, possibly, other COX defects.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmacy, Université de Montréal, Montréal, Canada.

ABSTRACT
Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC), a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase (COX) and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol), or modulate fatty acid (L-carnitine) or Krebs cycle metabolism (propionate) are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol) exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise questions about the nature of the diets, particularly excess fat intake, as well as on the use of antioxidants in patients with LSFC and, possibly, other COX defects.

No MeSH data available.


Related in: MedlinePlus

Effect of selected therapeutic agents on palmitate plus lactate (PL)-induced cytotoxicity.(A) Lactate dehydrogenase (LDH) release in control and LSFC fibroblasts exposed to PL in presence or absence of therapeutic compounds. The numbers in brackets (ex: control: n = 2:6) refer to the number of cell lines tested and of experiments, respectively. (B) Mean LDH release (n = 5), (C) Caspase 3/7 activity (n = 5), (D) Cellular ATP content (n = 7) and (E) COX/CS activity ratio (n = 3) measured after exposure to PL (palmitate 1 mM; lactate 10 mM) in absence or presence of CPMB (1 mM L-carnitine; 0.2 mM propionate; 125 nM Methylene blue) for 34 h (LDH release) or 24 h (other parameters). All measurements were performed in 4 control and 3 LSFC cell lines for the aforementioned number of experiments. Data are means ± S.E. and expressed as fold-change relative to the mean value of the PL condition in control (ctrl) cells. Statistical significance of differences between groups and conditions were analyzed with a linear hierarchical model (cf. Methods section). Significantly different from the control cells in the same experimental condition: * p < 0.05, ** p < 0.01. Significantly different from baseline within the same experimental group: ρp < 0.05, ρρp < 0.01, ρρρp < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4383560&req=5

pone.0120767.g004: Effect of selected therapeutic agents on palmitate plus lactate (PL)-induced cytotoxicity.(A) Lactate dehydrogenase (LDH) release in control and LSFC fibroblasts exposed to PL in presence or absence of therapeutic compounds. The numbers in brackets (ex: control: n = 2:6) refer to the number of cell lines tested and of experiments, respectively. (B) Mean LDH release (n = 5), (C) Caspase 3/7 activity (n = 5), (D) Cellular ATP content (n = 7) and (E) COX/CS activity ratio (n = 3) measured after exposure to PL (palmitate 1 mM; lactate 10 mM) in absence or presence of CPMB (1 mM L-carnitine; 0.2 mM propionate; 125 nM Methylene blue) for 34 h (LDH release) or 24 h (other parameters). All measurements were performed in 4 control and 3 LSFC cell lines for the aforementioned number of experiments. Data are means ± S.E. and expressed as fold-change relative to the mean value of the PL condition in control (ctrl) cells. Statistical significance of differences between groups and conditions were analyzed with a linear hierarchical model (cf. Methods section). Significantly different from the control cells in the same experimental condition: * p < 0.05, ** p < 0.01. Significantly different from baseline within the same experimental group: ρp < 0.05, ρρp < 0.01, ρρρp < 0.001.

Mentions: The effect of PL on LDH release, caspase 3/7 activities, ATP levels and COX activity was then examined in three LSFC and four control cell lines (see Table 1) and similar results were obtained (Fig. 4). The effect of PL on both LDH release and caspase 3/7 activities was also tested in the immortalized cell lines used to characterize baseline mitochondrial function. Although the effect occurred more rapidly in immortalized cells than in primary cells, LSFC fibroblasts still displayed an increased susceptibility to PL-induced cytotoxicity compared to their control counterparts (S2 Fig.).


Mitochondrial vulnerability and increased susceptibility to nutrient-induced cytotoxicity in fibroblasts from leigh syndrome French canadian patients.

Burelle Y, Bemeur C, Rivard ME, Thompson Legault J, Boucher G, LSFC ConsortiumMorin C, Coderre L, Des Rosiers C - PLoS ONE (2015)

Effect of selected therapeutic agents on palmitate plus lactate (PL)-induced cytotoxicity.(A) Lactate dehydrogenase (LDH) release in control and LSFC fibroblasts exposed to PL in presence or absence of therapeutic compounds. The numbers in brackets (ex: control: n = 2:6) refer to the number of cell lines tested and of experiments, respectively. (B) Mean LDH release (n = 5), (C) Caspase 3/7 activity (n = 5), (D) Cellular ATP content (n = 7) and (E) COX/CS activity ratio (n = 3) measured after exposure to PL (palmitate 1 mM; lactate 10 mM) in absence or presence of CPMB (1 mM L-carnitine; 0.2 mM propionate; 125 nM Methylene blue) for 34 h (LDH release) or 24 h (other parameters). All measurements were performed in 4 control and 3 LSFC cell lines for the aforementioned number of experiments. Data are means ± S.E. and expressed as fold-change relative to the mean value of the PL condition in control (ctrl) cells. Statistical significance of differences between groups and conditions were analyzed with a linear hierarchical model (cf. Methods section). Significantly different from the control cells in the same experimental condition: * p < 0.05, ** p < 0.01. Significantly different from baseline within the same experimental group: ρp < 0.05, ρρp < 0.01, ρρρp < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383560&req=5

pone.0120767.g004: Effect of selected therapeutic agents on palmitate plus lactate (PL)-induced cytotoxicity.(A) Lactate dehydrogenase (LDH) release in control and LSFC fibroblasts exposed to PL in presence or absence of therapeutic compounds. The numbers in brackets (ex: control: n = 2:6) refer to the number of cell lines tested and of experiments, respectively. (B) Mean LDH release (n = 5), (C) Caspase 3/7 activity (n = 5), (D) Cellular ATP content (n = 7) and (E) COX/CS activity ratio (n = 3) measured after exposure to PL (palmitate 1 mM; lactate 10 mM) in absence or presence of CPMB (1 mM L-carnitine; 0.2 mM propionate; 125 nM Methylene blue) for 34 h (LDH release) or 24 h (other parameters). All measurements were performed in 4 control and 3 LSFC cell lines for the aforementioned number of experiments. Data are means ± S.E. and expressed as fold-change relative to the mean value of the PL condition in control (ctrl) cells. Statistical significance of differences between groups and conditions were analyzed with a linear hierarchical model (cf. Methods section). Significantly different from the control cells in the same experimental condition: * p < 0.05, ** p < 0.01. Significantly different from baseline within the same experimental group: ρp < 0.05, ρρp < 0.01, ρρρp < 0.001.
Mentions: The effect of PL on LDH release, caspase 3/7 activities, ATP levels and COX activity was then examined in three LSFC and four control cell lines (see Table 1) and similar results were obtained (Fig. 4). The effect of PL on both LDH release and caspase 3/7 activities was also tested in the immortalized cell lines used to characterize baseline mitochondrial function. Although the effect occurred more rapidly in immortalized cells than in primary cells, LSFC fibroblasts still displayed an increased susceptibility to PL-induced cytotoxicity compared to their control counterparts (S2 Fig.).

Bottom Line: This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production.Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol), or modulate fatty acid (L-carnitine) or Krebs cycle metabolism (propionate) are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol) exacerbate palmitate plus lactate-induced cell death.Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise questions about the nature of the diets, particularly excess fat intake, as well as on the use of antioxidants in patients with LSFC and, possibly, other COX defects.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmacy, Université de Montréal, Montréal, Canada.

ABSTRACT
Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC), a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase (COX) and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol), or modulate fatty acid (L-carnitine) or Krebs cycle metabolism (propionate) are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol) exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise questions about the nature of the diets, particularly excess fat intake, as well as on the use of antioxidants in patients with LSFC and, possibly, other COX defects.

No MeSH data available.


Related in: MedlinePlus