Chronic expression of RCAN1-1L protein induces mitochondrial autophagy and metabolic shift from oxidative phosphorylation to glycolysis in neuronal cells

Abstract

Expression of the RCAN1 gene can be induced by multiple stresses. RCAN1 proteins (RCAN1s) have both protective and harmful effects and are implicated in common human pathologies. The mechanisms by which RCAN1s function, however, remain poorly understood. We identify RCAN1s as regulators of mitochondrial autophagy (mitophagy) and demonstrate that induction of RCAN1-1L can cause dramatic degradation of mitochondria. The mechanisms of such degradation involve the adenine nucleotide translocator and mitochondrial permeability transition pore opening. We also demonstrate that RCAN1-1L induction can shift cellular bioenergetics from aerobic respiration to glycolysis, yet RCAN1-1L has very little effect on cell division, whereas it has a cumulative negative effect on cell survival. These results shed the light on mechanisms by which RCAN1s can protect or harm cells and by which they may operate in human pathologies. They also suggest that RCAN1s are important players in autophagy and such elusive phenomena as the mitochondrial permeability transition pore.

FIGURE 1.

Mitochondrial mass evaluation in ST14A cells using FACS analysis. Cells were transfected with vector carrying RCAN1-1L fragment and with null vector (control). A, example of FACS analysis. Mitochondria were labeled with MitoTracker Green (Molecular Probes, Inc.), and signals were measured using the FACS analyzer (as described under “Experimental Procedures”). Mitochondrial mass was evaluated by comparison of the mean fluorescence signals between cells transfected with null vector and vector carrying RCAN1-1L. B, summary of mitochondrial mass FACS analysis. Dividing and differentiating cells were analyzed at the indicated time periods after transfection. Mean values were converted to arbitrary units (A.U.) to make the results from different experiments directly comparable with each other. Fluorescence values in samples transfected with null construct (control) were set as 1.0. Results (means ± S.E. (error bars)) represent measurements from three independent experiments. At the 6 h time point, mitochondrial mass was increased, whereas at all other time points it was significantly decreased, in cells overexpressing RCAN1-1L as compared with controls, as evaluated by Student's t test (p < 0.05).

FIGURE 2.

Electron microscopy analysis of mitochondria in ST14A cells. Cells were incubated to semiconfluence, and RCAN1-1L was overexpressed using our adenoviral construct. Dividing cells were collected in 24 h, whereas differentiating cells were collected 48 h after transfection. Cells were fixed and analyzed using transmission electron microscopy. A, representative photographs of cell sections (×5,000 magnification). Examples of mitochondria are marked by arrows. B, mitochondrial number. Whole cell sections were reconstructed, and, to ensure comparison only of cells cut close to the middle, only sections that spread into about three photomicrographs were analyzed. Twenty reconstructed cell sections were analyzed for each sample. Mitochondria were counted manually. In both cases, mitochondrial number was significantly decreased in cells overexpressing RCAN1-1L as compared with control cases, as evaluated by Student's t test (p < 0.05). C, mitochondrial size. Whole cell sections were reconstructed as in B, and the diameter of each mitochondrion was measured using a digital ruler (when mitochondria had an oval shape, diameter was calculated as (shortest diagonal + longest diagonal/2)). In differentiating cells, mitochondrial size was slightly (∼6%) but statistically significantly decreased in cells overexpressing RCAN1-1L as compared with control cases, as evaluated by Student's t test (p < 0.05). D, mitochondrial mass, in which the data from B and C were combined. Mitochondrial mass is ∼1.9 times reduced in dividing cells and ∼2.7 times reduced in differentiating cells after RCAN1-1L overexpression. This down-regulation is statistically significant at the p < 0.05 level, as evaluated by Student's t test. A.U., arbitrary units.

FIGURE 3.

RCAN1-1L-induced mitochondrial degradation involves activation of autophagy. A, levels of LC3 (I and II) are increased after RCAN1-1L overexpression. ST14A cells were transfected with vector carrying RCAN1-1L fragment or with null vector (C) for the indicated time periods. Control (C), in this particular case, represents cells transfected with null construct for 9 h. Signals were detected and quantified as described under “Experimental Procedures.” Probing with β-tubulin was used to control protein loadings. Results represent data from two separate experiments, in which two separate cell samples were used for each time point. Values shown are means ± S.E. (error bars) At 9 h, levels of LC3-II and LC3-I were markedly (∼2 times) increased in cells overexpressing RCAN1-1L as compared with controls as evaluated by Student's t test (p < 0.05). B, autophagic flux after RCAN1-1L overexpression. Cells were transfected, and at the 6 h time point after transfection, they were incubated for another 3 h (total 9 h after transfection) either with (Inhib+) or without (Inhib−) inhibitors of lysosomal proteolysis (20 mm NH₄Cl + 100 μm leupeptin) to prevent autophagosome clearance. Levels of LC3-II were measured as in A. Three separate cell samples were used for each time point. Values shown are means ± S.E. C, the number of autophagosomes is increased after RCAN1-1L overexpression. Dividing ST14A cells were collected at 24 h after RCAN1-1L overexpression and analyzed using TEM as shown in supplemental Fig. 2. Whole cell sections were reconstructed, and, to ensure comparison only of cells cut close to the middle, only sections that spread into at least three photomicrographs were analyzed. Ten reconstructed cell sections were analyzed for each sample. Autophagosomes were counted manually. Autophagosome number was significantly (∼2.2-fold) increased in cells overexpressing RCAN1-1L in comparison with control cells, as evaluated by Student's t test (p < 0.05). A.U., arbitrary units.

FIGURE 4.

mTOR signaling and expression of PGC-1α after RCAN1-1L overexpression. ST14A cells were transfected with vector carrying RCAN1-1L fragment and with null vector (C). Signals were detected and quantified as described under “Experimental Procedures.” p70S6K(Thr-389) was detected as a ∼68-kDa size protein. The expected ∼113-kDa size PGC-1α protein is marked by an arrow. Probing with β-tubulin was used to control protein loading. Results represent data from two separate experiments, in which three separate cell samples were used for each time point. Values shown are means ± S.E. (error bars). A, 9 h after RCAN1-1L overexpression. Levels of p70S6K(Thr-389) were significantly (∼75% or ∼3-fold) decreased in cells overexpressing RCAN1-1L as compared with controls, as evaluated by Student's t test (p < 0.05). Levels of PGC-1α were significantly (62%) increased in cells overexpressing RCAN1-1L as compared with controls, as evaluated by Student's t test (p < 0.05). B, 24 h after RCAN1-1L overexpression. Levels of both p70S6K(Thr-389) and PGC-1α were significantly (46 and 38%, respectively) decreased in cells overexpressing RCAN1-1L compared with controls, as evaluated by Student's t test (p < 0.05). A.U., arbitrary units.

FIGURE 5.

mtPTP dynamics in dividing ST14A cells after RCAN1-1L overexpression. Cells were transfected with vector carrying RCAN1-1L fragment and with null vector (control). A, example of FACS analysis. Assays were performed in intact cells using the calcein fluorescence dye as described under “Results.” B, summary of PTP analysis. Cells were analyzed at the indicated time periods after transfection. Mean values were converted to arbitrary units (A.U.) to make the results from different experiments directly comparable with each other. Fluorescence values in samples transfected with null construct (control) were set as 1.0. Results (means ± S.E. (error bars)) represent measurements from three independent experiments. Obtained numbers represent mtPTP values per cell, which also were expressed per mitochondrion, taking into account data shown in Figs. 2 and 3. At the 3 and 6 h time points after RCAN1-1L overexpression, mtPTP values were significantly decreased either per cell or per mitochondrion (Student's t test, p < 0.05). At the 12 and 24 h time points, mtPTP values were significantly increased either per cell or per mitochondrion (Student's t test, p < 0.05). At the 72 h time point after RCAN1-1L overexpression, mtPTP values become equal as expressed per cell; however, they remained elevated when expressed per mitochondrion (Student's t test, p < 0.05).

FIGURE 6.

RCAN1-1L reduces OCR but increases ECAR. RCAN1-1L was overexpressed using our adenoviral construct. Dividing cells were analyzed 24 h after transfection, and differentiating cells were analyzed 48 h after transfection. A, example of real-time analysis of bioenergetic pathways in ST14A cells. OCR and ECAR were monitored over time using the Seahorse metabolic analyzer. Cells were grown in 24-well plates to semiconfluence. To control cell amount, total protein content was determined in each well after the measurements, and data were normalized relative to protein content. Cells were incubated with oligomycin, FCCP, and rotenone. Results are means ± S.E. (error bars) of four independent samples. B, OCR. Values before the addition of oligomycin were used to calculate basal mitochondrial respiration. Values in the presence of FCCP were used to calculate maximal mitochondrial respiration. Results represent measurements from three experiments ± S.E. Both basal and maximal OCR were significantly reduced after RCAN1-1L overexpression in dividing as well as differentiating cells, as evaluated by Student's t test (p < 0.05). C, ECAR. Results represent measurements from three experiments ± S.E. ECAR were significantly increased after RCAN1-1L overexpression in both dividing and differentiating cells, as evaluated by Student's t test (p < 0.05). D, basal OCR/ECAR ratio. Results represent measurements from three experiments ± S.E. The ratios were significantly decreased after RCAN1-1L overexpression in both dividing and differentiating cells evaluated by Student's t test (p < 0.05). E, ATP levels are decreased after RCAN1-1L overexpression. Dividing ST14A cells were collected at 24 h after RCAN1-1L overexpression and analyzed as described under “Experimental Procedures.” ATP levels were significantly (∼40%) decreased in cells overexpressing RCAN1-1L in comparison with control cells, as evaluated by Student's t test (p < 0.05).

FIGURE 7.

Effect of RCAN1-1L overexpression on cell division and survival. A, dividing ST14A Cells. Cell number was measured using a Coulter Counter (Coulter Corp.). The results represent mean values of three independent experiments ± S.E. (error bars). After 72 h, the number of cells in the samples overexpressing RCAN1-1L was significantly (∼17%) lower than in controls as evaluated by Student's t test (p < 0.05). B, dividing ENStem cells. Cell number was measured using a Coulter Counter. The results represent mean values of three independent experiments ± S.E. After 72 h, the number of cells in the samples overexpressing RCAN1-1L was significantly (∼10%) lower than in controls, as evaluated by Student's t test (p < 0.05). C, differentiating ST14A cells. Cells were analyzed microscopically and counted manually. Only live differentiating cells were counted. The results represent the means of three independent experiments ± S.E. After 72 h, the number of cells in the samples overexpressing RCAN1-1L was significantly (∼17%) lower than in controls, as evaluated by Student's t test (p < 0.05). D, differentiating ENStem cells. Cells were grown and differentiated for 12 days (288 h). They were then analyzed microscopically and counted manually. Only live differentiating cells were counted. The results represent the means of three independent experiments ± S.E. The number of cells in the samples overexpressing RCAN1-1L was significantly (∼25%) lower than in controls, as evaluated by Student's t test (p < 0.05).