Mitochondrial function and morphology are impaired in parkin-mutant fibroblasts

Abstract

Objective: There are marked mitochondrial abnormalities in parkin-knock-out Drosophila and other model systems. The aim of our study was to determine mitochondrial function and morphology in parkin-mutant patients. We also investigated whether pharmacological rescue of impaired mitochondrial function may be possible in parkin-mutant human tissue.

Methods: We used three sets of techniques, namely, biochemical measurements of mitochondrial function, quantitative morphology, and live cell imaging of functional connectivity to assess the mitochondrial respiratory chain, the outer shape and connectivity of the mitochondria, and their functional inner connectivity in fibroblasts from patients with homozygous or compound heterozygous parkin mutations.

Results: Parkin-mutant cells had lower mitochondrial complex I activity and complex I-linked adenosine triphosphate production, which correlated with a greater degree of mitochondrial branching, suggesting that the functional and morphological effects of parkin are related. Knockdown of parkin in control fibroblasts confirmed that parkin deficiency is sufficient to explain these mitochondrial effects. In contrast, 50% knockdown of parkin, mimicking haploinsufficiency in human patient tissue, did not result in impaired mitochondrial function or morphology. Fluorescence recovery after photobleaching assays demonstrated a lower level of functional connectivity of the mitochondrial matrix, which further worsened after rotenone exposure. Treatment with experimental neuroprotective compounds resulted in a rescue of the mitochondrial membrane potential.

Interpretation: Our study demonstrates marked abnormalities of mitochondrial function and morphology in parkin-mutant patients and provides proof-of-principle data for the potential usefulness of this new model system as a tool to screen for disease-modifying compounds in genetically homogenous parkinsonian disorders.

Figure 1

Mitochondrial respiratory chain function in controls and parkin mutant fibroblasts. (A) Overall mitochondrial membrane potential in the patient group is decreased by 30%, ** p < 0.01 when cells are grown in glucose containing culture medium, however this reduction is even more severe (reduction by 70%, *** p < 0.0001) in the patient group when cells are grown in galactose containing medium. Measurement of mitochondrial membrane potential was performed on 3 separate occasions in all samples, data is presented as mean +/- SEM. All subsequent biochemical and morphological analyses were also performed on 3 separate occasions and are presented as mean +/- SEM unless otherwise stated. (B) Subsequent spectrophotometric assessment of individual respiratory chain complexes demonstrated a specific reduction of complex I activity in the patient group by 45%, ** p < 0.01. (C) In contrast, complex II activity was similar in patients and controls, p > 0.05.

Figure 2

ATP production in controls and parkin mutant fibroblasts. (A) Complex I linked ATP production was reduced in the patient group by 48%, * p < 0.05. (B) In contrast, complex II linked ATP production was not significantly altered between the control and patient groups. (C) The reduction in the complex I linked ATP production also led to an overall reduction in cellular ATP levels by 58%, * p < 0.05.

Figure 3

Mitochondrial morphology in control and parkin mutant fibroblasts. (A) Images of mitochondria in control and patient fibroblasts, illustrating the increase in mitochondrial branching in patient cells. (B) Mitochondrial branching (form factor) is significantly increased in the patient group, * p < 0.05. Images of 25 individual cells were assessed per cell line per day and this was repeated on 3 separate occasions. Data is presented in box and whisker plot with SEM and mean. All subsequent morphological analysis was performed and is presented in the same way. (C) In contrast, mitochondrial length (aspect ratio) is similar in the control and patient group. (D) Mitochondrial number per cell is decreased by 20%, but this did not reach statistical significance, p > 0.05. (E+F) In individual patients complex I linked ATP production correlates with both mitochondrial branching (form factor, R² = 0.903, p < 0.0001) and mitochondrial length (aspect ratio, R² = 0.58, p < 0.05). (G) Changes in mitochondrial branching (form factor) correlate with complex I activity as well (R² = 0.632, p < 0.01).

Figure 4

siRNA mediated knockdown of parkin in control fibroblasts. (A) Western blot of actin and parkin protein levels. Parkin protein levels are reduced by 80% after siRNA transfection. There was no change in parkin or actin protein levels in either scramble siRNA or GAPDH siRNA transfected cells. (B) siRNA knockdown of GAPDH efficiency assessed by western blotting of GAPDH protein and quantified using densitometry, showed a 70% reduction in GAPDH protein levels. (C) This siRNA mediated knockdown of parkin resulted in a decrease of the mitochondrial membrane potential by 80%, ** p < 0.01, with no effect of either scramble or GAPDH siRNA. (D) Cellular ATP levels are also reduced by 42%, ** p < 0.01, with no effect of either scramble or GAPDH siRNA. (D) Mitochondrial branching (form factor, FF) is significantly increased in siRNA parkin cells ** p < 0.01. In contrast, mitochondrial length (aspect ratio, AR) and mitochondrial number per cell (Nc) are similar in cells transfected with scramble siRNA, GAPDH siRNA or parkin siRNA. (E) 50% knockdown of parkin does not result in a change of mitochondrial membrane potential. (F) There is also no effect of 50% parkin knockdown on length (aspect ratio), branching (form factor) or number of mitochondria per cell.

Figure 5

The effect of rotenone treatment on control and parkin mutant fibroblasts. (A) Rotenone treatment leads to a reduction of the mitochondrial membrane potential in control cells by 54%, * p < 0.05. There was no further reduction of the already markedly lower mitochondrial membrane potential in parkin mutant cells after rotenone exposure. (B) Images of fibroblasts from parkin mutant patients before and after rotenone treatment, illustrating the reduction of absolute length of the mitochondria after toxin exposure (see also Fig 5B). (C) Mitochondrial length (aspect ratio) is shortened by 42% in parkin mutant cells after rotenone exposure, ** p = 0.01. Similar shortening of the mitochondria is observed in cells with siRNA mediated parkin knockdown after rotenone exposure, ** p = 0.01. In contrast, mitochondrial length in control fibroblasts remains unchanged after rotenone exposure. (D) Mitochondrial branching (form factor) is increased in control and patient cells after rotenone exposure (* p < 0.05) to a similar extent observed in siRNA-mediated parkin knockdown cells, which showed no further increase in branching after rotenone exposure.

Figure 6

Mitochondrial fission induced by rotenone is enhaned by parkin deficiency. (A) Fluoresence recovery after photobleaching (FRAP) curves in controls (n=4; open symbols) and parkin deficient cell lines (n=5 closed symbols) either without treatment (circles) or after exposure to rotenone (squares). Each point is the fluorescence intensity of mito-YFP, normalized to prebleach at the times indicated on the x-axis. (B) The fraction of mito-YFP that is mobile (mobile fraction, y-axis) is lower in parkin deficient lines (filled bars) than in controls (open bars: * p < 0.05); rotenone enhances these effects (* p < 0.05). (C) The basal defect in mobile fraction in the parkin deficient lines (filled symbols) compared to controls (open symbols) correlates with lower complex I activity (r=0.79, P=0.01). where n=3 for complex I activity and n=27-30 for mobile fraction.

Figure 7

Rescue of the mitochondrial membrane potential by experimental neuroprotective compounds. (A) Concentration response curve measuring mitochondrial membrane potential in 3 different parkin mutant fibroblasts lines treated with 2-oxo-4-thiazolidine carboxylic acid (OTCA). The horizontal line marks the control values for mitochondrial membrane potential. (B) The mitochondrial membrane potential is nearly normalized (95% of controls) with treatment of OTCA. Treatment with glutathione methyl ester (GME) results in less marked (65% of controls), but still significant increase, ** p < 0.01. (C) Complex I activity is not increased after treatment with OTCA. (D) In contrast, complex II activity is increased after treatment with -oxo-4-thiazolidine carboxylic acid (OTCA) in patient cells * p < 0.05. (E) Cellular ATP levels after treatment with OTCA are significantly increased in patient cells ** p < 0.01.