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. 2019 Oct 17:13:1070.
doi: 10.3389/fnins.2019.01070. eCollection 2019.

ASCs-Exosomes Recover Coupling Efficiency and Mitochondrial Membrane Potential in an in vitro Model of ALS

Elisa Calabria  1 , Ilaria Scambi  1 , Roberta Bonafede  1 , Lorenzo Schiaffino  1 , Daniele Peroni  2 , Valentina Potrich  2 , Carlo Capelli  1   3 , Federico Schena  1 , Raffaella Mariotti  1
Affiliations

ASCs-Exosomes Recover Coupling Efficiency and Mitochondrial Membrane Potential in an in vitro Model of ALS

Elisa Calabria et al. Front Neurosci. .

Abstract

The amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneurons death. Mutations in the superoxide dismutase 1 (SOD1) protein have been identified to be related to the disease. Beyond the different altered pathways, the mitochondrial dysfunction is one of the major features that leads to the selective death of motoneurons in ALS. The NSC-34 cell line, overexpressing human SOD1(G93A) mutant protein [NSC-34(G93A)], is considered an optimal in vitro model to study ALS. Here we investigated the energy metabolism in NSC-34(G93A) cells and in particular the effect of the mutated SOD1(G93A) protein on the mitochondrial respiratory capacity (complexes I-IV) by high resolution respirometry (HRR) and cytofluorimetry. We demonstrated that NSC-34(G93A) cells show a reduced mitochondrial oxidative capacity. In particular, we found significant impairment of the complex I-linked oxidative phosphorylation, reduced efficiency of the electron transfer system (ETS) associated with a higher rate of dissipative respiration, and a lower membrane potential. In order to rescue the effect of the mutated SOD1 gene on mitochondria impairment, we evaluated the efficacy of the exosomes, isolated from adipose-derived stem cells, administrated on the NSC-34(G93A) cells. These data show that ASCs-exosomes are able to restore complex I activity, coupling efficiency and mitochondrial membrane potential. Our results improve the knowledge about mitochondrial bioenergetic defects directly associated with the SOD1(G93A) mutation, and prove the efficacy of adipose-derived stem cells exosomes to rescue the function of mitochondria, indicating that these vesicles could represent a valuable approach to target mitochondrial dysfunction in ALS.

Keywords: ALS; NSC-34 cell line; complex I; coupling efficiency; exosomes; high resolution respirometry; membrane potential; mitochondria.

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Figures

FIGURE 1
FIGURE 1
Oxygen consumption and flux control ratios of the naïve NSC-34 intact cells obtained by high resolution respirometry. (A) Rate of oxygen consumption of intact naïve NSC-34 measured at 37°C in DMEM in the following states: the ROUTINE respiration sustained by pyruvate, malate, and endogenous substrates; the LEAK respiration obtained following addition of olygomicin; the maximal uncoupled ETS capacity (addition of CCCP) and the ROX, residual oxygen consumption following addition of the inhibitor of complex III antimycin A. The red line represent the rate of oxygen consumption normalized per number of cells. (B) Quantitative analysis of data of oxygen consumption in the different respiratory states. (C) Flux control ratios, data normalized to the ETS. Data in (B,C) are represented as mean ± SD, n = 7.
FIGURE 2
FIGURE 2
The expression of the human mutant SOD1(G93A) in NSC-34 cells impairs mitochondrial function. (A) Representative Western blot assay performed on cells lysates of WTDOXY–, G93ADOXY–, G93ADOXY+, and WTDOXY+ cells. Endogenous mouse SOD1 (mSOD1) immunoreactivity was detected in all samples with anti-SOD1 antibody (left panel), whereas HA-tagged human SOD1 (hSOD1) was detected only in (DOXY+) samples following incubation for 24 h with doxycycline (right panel), n = 3. (B) Mitochondrial flux control ratios of naïve, WTDOXY+, G93ADOXY–, and G93ADOXY+ intact cells in the LEAK state relative to maximal ETS capacity. (C) ETS coupling efficiency of naïve, WTDOXY+, G93ADOXY–, and G93ADOXY+ intact cells determined as 1-(L/E). One-way ANOVA analysis; ∗p < 0.05, ∗∗p < 0.01. Data are represented as mean ± SD, n = 4.
FIGURE 3
FIGURE 3
Mitochondrial complexes contribution to oxidative phosphorylation in permeabilized G93ADOXY+ and G93ADOXY– cells. (A) Rate of oxygen consumption (red line) of permeabilized G93ADOXY– cells measured at 37°C in Mir05 in the following states: the ROUTINE respiration sustained by pyruvate and malate (PM) and endogenous substrates; the LEAK respiration evaluated following permeabilization with digitonin; the ADP addition stimulated OXHPOS capacity sustained by complex I (pyruvate, malate and glutamate, PMG) and complex II (pyruvate, malate, glutamate and succinate, PMGS); the maximal uncoupled ETS capacity (addition of CCCP); ETS sustained by complex II following inhibition of complex I by rotenone; the ROX, residual oxygen consumption, following addition of the inhibitor of complex III antimycin A. (B–E) Dot plots corresponding to respirometry experiments, showing data from all samples. Each dot corresponds to one sample. The line shows the mean with standard deviation. Paired samples are represented by symbols of the same color. (B) Quantitative analysis of the rate of oxygen consumption of G93ADOXY– and G93ADOXY+ permeabilized cells: the values relative to ROUTINE, LEAK, OXPHOS, and ETS for paired samples are reported. (C–E) Flux control ratios, normalized to the maximal uncoupled ETS capacity, of G93ADOXY–, and G93ADOXY+ permeabilized cells in the LEAK, OXPHOS and ETS(cII) respiratory states. Data are represented as percentage of control (G93ADOXY– cells). Data of flux control ratios (mean ± SD) in G93ADOXY– cells are: (C) LEAK (0.28 ± 0.03), (D) OXPHOS(PMG) (0.50 ± 0.04), and (E) ETS(cII) (0.69 ± 0.05). Analysis of G93ADOXY– vs. G93ADOXY+ cells at various respiratory states was performed using two-way ANOVA (Holm-sidak’s post hoc correction) ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (B); paired student T test (C–E) ∗p < 0.05. n = 9.
FIGURE 4
FIGURE 4
TEM and Western blot analysis of ASCs-exosomes. (A) Electron microscopy shows vesicles with characteristic morphology and size of exosomes. Scale bar, 200 nm. (B) The blots show Western blot detection of the expression of Alix (90 kDa), CD81 (26 kDa), and HSP70 (70 kDa) in exosomes (EXO); ASCs lysates were used as positive control.
FIGURE 5
FIGURE 5
Mitochondrial function in G93ADOXY+ cells after exosomes treatment. Dot plots of data obtained from respirometry experiments, showing the effects of vehicle (PBS), or exosomes (EXO) treatment in DOXY+ cells for all samples. Each dot corresponds to one sample. The line shows the mean with standard deviation. Paired samples are represented by symbols of the same color. (A) ETS coupling efficiency in G93ADOXY+ intact cells following treatment with exosomes (EXO) or PBS. (B) Flux control ratio of the OXPHOS sustained by complex I (PMG) in G93ADOXY+ permeabilized cells following incubation with exosomes (EXO) or PBS. (C) Flux control ratio of the OXPHOS sustained by complex II (PMGS) in G93ADOXY+ permeabilized cells following incubation with exosomes (EXO) or PBS. Comparison of EXO vs. PBS treated G93ADOXY+ cells was performed using paired t-test. Data are represented as percentage of control (PBS). Data of flux control ratios (mean ± SD) in PBS treated cells cells are: (A) ETS coupling efficiency (0.54 ± 0.13), (B) OXPHOS(PMG) (0.29 ± 0.07), and (C) OXPHOS(PMGS) (0.81 ± 0.12). Paired T test ∗p < 0.05, ∗∗p < 0.01. n = 7.
FIGURE 6
FIGURE 6
Flow cytometry for mitochondrial membrane potential evaluation in G93ADOXY+ and G93ADOXY– cells. (A,B) Representative dot plots of flow cytometry of all events collected using gates to exclude dead cells and debris (A) and to select the singlets (single cells) (B). Cell size (forward scatter, FSC) vs. cellular granularity (side scatter, SSC) data are plotted. (C) The histograms show the intensity of DiOC6(3) probe in the G93ADOXY– cells, G93ADOXY+ cells, and G93ADOXY+ cells treated with exosomes (EXO). (D) Measurement of mitochondrial membrane potential with DiOC6(3) probe in cells G93ADOXY– and G93ADOXY+ following treatment with exosomes. Data are reported as median fluorescence intensity (MFI) of the probe relative to the G93ADOXY– cells. The analysis was performed using one-way ANOVA with Tukey correction; p < 0.05. Data are represented as mean ± SD, ∗p < 0.05, ∗∗p < 0.01. n = 9.
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