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. 2012 Jun;1822(6):1019-29.
doi: 10.1016/j.bbadis.2012.01.010. Epub 2012 Jan 28.

Metabolically induced heteroplasmy shifting and l-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS

Valerie Desquiret-Dumas  1 , Naig Gueguen, Magalie Barth, Arnaud Chevrollier, Saege Hancock, Douglas C Wallace, Patrizia Amati-Bonneau, Daniel Henrion, Dominique Bonneau, Pascal Reynier, Vincent Procaccio
Affiliations

Metabolically induced heteroplasmy shifting and l-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS

Valerie Desquiret-Dumas et al. Biochim Biophys Acta. 2012 Jun.

Abstract

The m.3243A>G variant in the mitochondrial tRNA(Leu(UUR)) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood. We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A>G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition of l-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition of l-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting and l-arginine therapy may constitute promising therapeutic strategies against MELAS.

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Figures

Figure 1
Figure 1
Cellular metabolism of the m.3243A>G SH-SY5Y cybrid cells. (A) Glucose consumption, pyruvate, lactate and alanine production in controls (open bars), 70% M cybrids (grey bars) and 100% M cybrids (black bars). (B) Enzymatic activity of lactate dehydrogenase in controls (open bars), 70% M cybrids (grey bars) and 100% M cybrids (black bars). The results are expressed as fold changes relative to mean control cell values. Data are presented as means ± sem of 4 independent experiments. Differences between groups were evaluated by a Mann-Whitney statistical test, the asterisk (*) indicates significant differences (p<0.05) compared to controls.
Figure 2
Figure 2
Oxygen consumption assays on cybrid cells. (A) Mitochondrial oxygen consumption measured on digitonin-permeabilized controls (open bars), 70% M cybrids (grey bars) and 100% M cybrids (black bars). The results show complex I, complex II and complex IV-dependent respiratory rates. (B) Total ATP content of controls (open bars), 70% M cybrids (grey bars) and 100% M cybrids (black bars). The results are expressed as fold changes relative to mean control cell values. The data are presented as means ± sem of 4 independent experiments. Differences between groups were evaluated by a Mann-Whitney statistical test, the asterisk (*) indicates significant differences (p<0.05) compared to controls.
Figure 3
Figure 3
The m.3243A>G mutation affects the structure of the mitochondrial network. Control SHSY5Y, 70% M cybrids and 100% M cybrids were cultured on chamber-slides (Lab-Tek). Mitochondria were labelled with MitoTracker® Green 100 nM, 15min in DMEM-High Glucose, 10% FBS. Representative images are shown here. A colour code indicates the different types of mitochondria. Connected mitochondrial networks are shown in purple/blue whereas isolated and spherical mitochondria are shown in yellow/red. The histogram shows the percentage of connected or unconnected mitochondria per cell line. The results are the means of two independent experiments.
Figure 4
Figure 4
Respiratory chain complex activities and assembly of the cybrid cells. (A) Maximal activity of complexes I, II and IV of controls (open bars), 70% M cybrids (grey bars) and 100% M cybrids (black bars). (B) Representative western blot analysis of subunits of Complex I (20 kDa), II (30 kDa), III (core 2), IV (COXII, mtDNA-encoded and COX IV, nuclear DNA-encoded) and V (F1α) and of a reference loading protein (α-tubulin). (C) Blue-Native PAGE analysis of the respiratory chain complex assembly of cybrid cells. Holoenzyme complexes and sub-complexes were visualized by enhanced chemiluminescence after primary antibody incubation (NDUFB6 for Complex I, COX I for Complex IV and II 70 kDa for Complex II used as loading reference).
Figure 5
Figure 5
Oxidative stress measurements in cybrid cells. (A) The concentration of nitrates + nitrites concentration in the supernatant of controls (open bars), 70% M cybrids (grey bars) and 100% M cybrids (black bars). (B) GSH concentration in the supernatant of controls (open bars), 70% M cybrids (grey bars) and 100% M cybrids (black bars). (C) Western Blot analysis of antioxidant mitochondrial MnSOD. The beta subunit of complex V (Vbeta) was used as loading reference. (D) Ratio of aconitase/citrate synthase activity in controls (open bars), 70% M cybrids (grey bars) and 100% M cybrids (black bars). The results are expressed as fold changes relative to means of control cell values. The data are presented as means ± sem of 4 independent experiments. Differences between groups were evaluated by a Mann-Whitney statistical test; the asterisk (*) indicates significant differences (p<0.05) compared with controls.
Figure 6
Figure 6
Mitochondrial respiratory chain enzyme assays and assembly of the arginine-treated cybrid cells. (A) The ratio of complex I/citrate synthase in controls (open bars), and in arginine-treated 70% M cybrids (grey bars) and 100% M cybrids (black bars). (B) The ratio of complex IV/citrate synthase in controls (open bars), and in arginine-treated 70% M cybrids (grey bars) and 100% M cybrids (black bars). The results are expressed as fold changes relative to means of control cell values. The data are presented as means ± sem of 4 independent experiments. Differences between groups were evaluated by a Mann-Whitney statistical test; the asterisk (*) indicates significant differences (p<0.05) compared with controls and the sharp sign (#) indicates significant differences between treated and untreated cells (p<0.05). (C) Blue-Native PAGE analysis of complex I assembly in arginine-treated cybrid cells. The holoenzyme complexes and sub-complexes were visualized by enhanced chemiluminescence after primary antibody incubation (NDUFB6). (D) Blue-Native PAGE analysis of complex IV assembly of arginine-treated cybrids (COX I antibody).
Figure 7
Figure 7
Blue-Native PAGE analysis of respiratory chain complex assembly of 90% M cybrids compared to 70% and 100% M cybrid cells and control cells. All cell lines were cultured in the same media. Holoenzyme complexes and sub-complexes were visualized by enhanced chemiluminescence after primary antibody incubation (NDUFB6 for complex I and COX I for complex IV).
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