Functional mitochondrial impairments in fibroblasts with parkin mutations
We first investigated the mitochondrial membrane potential in primary fibroblast cultures of 5 patients with homozygous or compound heterozygous parkin mutations (see ) and 6 age matched controls to determine the overall oxidative phosphorylation activity of the mitochondrial respiratory chain in parkin mutant patient tissue. Mitochondrial membrane potential was lower in all 5 parkin mutant fibroblast cultures by an average of 30% (; p < 0.01). However, when the culture medium was changed to include galatcose as an energy source rather than glucose, thereby switching it to a more oxidative state, the mitochondrial membrane potential was then even lower by an average of 70% (; p < 0.0001).
Parkin mutations detected on either allele in the five patients with early onset parkinsonism and homozygous or compound heterozygous parkin mutations. See method section for further details.
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 (more ...)
We next determined whether the observed decrease in the mitochondrial membrane potential was related to impaired function of a particular complex of the mitochondrial respiratory chain or due to overall impaired function. Spectrophotometric assessment of the activity of each individual complex in mitochondrially enriched fractions demonstrated that complex I activity was lower by an average of 42% in parkin mutant fibroblasts compared to controls (; p < 0.005). Complex II activity showed a trend towards higher activity, but this was not significant (; p > 0.05). Complex III and complex IV activity were similar in the parkin mutant patients and controls (data not shown).
We next determined whether the observed complex I deficiency leads to overall impaired mitochondrial ATP production or to impairment of complex I-linked ATP production only. ATP production was lower after specific substrates (glutamate and malate) for complex I (p < 0.05; ), but not complex II (succinate) were added (). The lower complex I-linked ATP production also results in an overall decrease of cellular ATP levels by 58% in the parkin mutant fibroblasts (; 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 (more ...)
Mitochondrial morphology is significantly altered in parkin mutant fibroblasts
Qualitative assessment in individual parkin mutant patients revealed an at times rather marked increase in mitochondrial branching () which was confirmed using quantitative morphological analysis of the parkin mutant patient cohort (form factor, p < 0.05, ), without a significant group change in mitochondrial length (aspect ratio, p > 0.05, ). The overall number of mitochondria showed a trend towards lower numbers in the parkin mutant fibroblasts, but this did not reach statistical significance (p > 0.05, ). The lower complex I linked ATP production correlates with an increase in both the degree of mitochondrial branching (form factor, R2 = 0.903, p < 0.001; ) and length (aspect ratio, R2 = 0.58, p < 0.05, ). In addition an increase in the degree of mitochondrial branching (form factor) correlates with lower complex I activity (R2 = 0.632. p < 0.01; ). This indicates that the functional and morphological effects of parkin deficiency on mitochondria are related to each other.
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 (more ...)
Parkin siRNA knockdown confirms impaired mitochondrial respiratory chain function and morphology in parkin deficiency
Parkin protein levels were reduced by 80% in siRNA treated control fibroblasts compared to mock transfected controls at 5 days post transfection (). Mitochondrial membrane potential was significantly lower in siRNA-mediated parkin knockdown cells at 5 days post transfection (, p < 0.01). Cellular ATP levels were also significantly lower in siRNA-mediated parkin knockdown cells at 5 days post transfection (, p < 0.01). Parkin protein levels, mitochondrial membrane potential and cellular ATP levels returned back to control levels 9 days post transfection (data not shown).
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 (more ...)
We next investigated whether the changes in mitochondrial morphology observed in fibroblasts from patients with parkin mutations were also present in parkin knockdown fibroblasts. Mitochondrial branching was significantly increased by 70% (, p < 0.01) in siRNA mediated parkin knockdown fibroblasts at 5 days. In contrast, there were no significant changes in mitochondrial length and number in the parkin knockdown fibroblasts. There was no effect of either scramble siRNA or GAPDH siRNA on any of the parameters measured. The siRNA mediated parkin knockdown data therefore show that parkin deficiency is sufficient to explain the mitochondrial deficiencies seen in the patient fibroblasts. In contrast, 50% knockdown of parkin, mimicking haploinsufficiency in human patient tissue, did not result in impaired mitochondrial transmembrane potential or alteration of mitochondrial morphology ().
Parkin mutant fibroblasts are more susceptible to exposure to mitochondrial toxins
-mutant fibroblasts and control fibroblasts were then exposed to the mitochondrial complex I inhibitor rotenone to determine whether endogenous parkin has a protective effect against the effect of rotenone on mitochondrial membrane potential and mitochondrial morphology in human tissue.16
Mitochondrial membrane potential was decreased in control fibroblasts exposed to rotenone by 52%, but not in parkin
-mutant fibroblasts or cells with siRNA-mediated parkin
knockdown (p < 0.05; ). This could indicate a floor effect of our experimental protocol or that the mitochondrial membrane potential is already maximally reduced by the existing complex I defect in the parkin
-mutant fibroblasts (see also ).
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 (more ...)
Mitochondrial morphology was altered in cells exposed to rotenone, with a tendency to form short, irregularly shaped mitochondria suggesting increased fission, especially in the patient fibroblasts (). To quantify these effects, we measured branching (form factor) and length (aspect ratio) in patient and control cells under basal and rotenone conditions. Rotenone exposure resulted in significantly decreased mitochondrial length in parkin-mutant fibroblasts, but not in controls (aspect ratio, p < 0.01, ). The reduction in mitochondrial length after rotenone exposure was confirmed in fibroblasts with siRNA mediated knockdown of parkin (p < 0.02, ).
Mitochondrial branching was increased by 50% in control fibroblasts exposed to rotenone (form factor, p < 0.02, ), resembling the extent of branching observed in mutant parkin fibroblasts under normal conditions. Mutant parkin fibroblasts also have significantly increased mitochondrial branching as compared to before rotenone treatment (, p < 0.02). As described above, there was already markedly increased mitochondrial branching after siRNA-mediated parkin knock down (see ), comparable to the extent of branching observed in parkin mutant cells after rotenone exposure (). There was no further increase in branching after rotenone exposure in the siRNA-mediated parkin knockdown cells.
Additive effect of parkin mutations and pharmacological complex I inhibition on mitochondrial functional connectivity
The above results suggest that parkin deficient fibroblasts have morphological alterations that are worsened by exposure to additional stressors such as rotenone. We next used the FRAP assay to further determine whether there is impairment of functional connectivity in the parkin-mutant cells. Quantification of FRAP showed that parkin deficient cells had lower recovery of mito-YFP than controls under basal conditions and even more so after rotenone exposure (). To provide an overall summary measure of the FRAP results, we calculated the mobile fraction of mito-YFP over time (). Using this as a metric, the effect of genotype and rotenone were both significant (two way ANOVA, Pgenotype=0.0036, Protenone=0.006). The mobile fraction values correlated with complex I activity in the cells (; r=0.79, P=0.01). Overall, these results show that parkin deficient fibroblasts have a basal defect in functional connectivity that is enhanced by rotenone.
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) (more ...)
Mitochondrial respiratory chain defect can be rescued in the parkin mutant fibroblasts by treatment with glutathione replacement compounds
Control and parkin-mutant fibroblasts were exposed to the glutathione precursor OTCA. A concentration response curve was performed using 24 hour treatment with OTCA (). Complete recovery of mitochondrial membrane potential in parkin deficient cells was achieved with the highest dose of OTCA used (30μM). The rescue effect was concentration dependent with a calculated EC90 of 10μM which was used in all subsequent experiments.
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 (more ...)
Treatment of all 5 parkin mutant fibroblasts with 10μM OTCA showed improvements of mitochondrial membrane potential in every individual patient, with the average membrane potential being restored to 95% of control levels (). In contrast, treatment with GME at a dose of 100μM increased the mitochondrial membrane potential to 60% of control values but could not completely replenish mitochondrial membrane potential (). Similar results were obtained with higher doses of GME (data not shown). These data indicate that the observed near-complete rescue effect with OTCA may only partially be due to its effect on intracellular glutathione levels. In order to investigate the underlying mechanism of this rescue effect on the mitochondrial membrane potential by OTCA further in depth experiments were performed. Incubation of cells with 10μM OTCA did not alter mitochondrial complex I activity (). In contrast, complex II activity was significantly increased (). Cellular ATP levels were completely restored in all patients after treatment with 10μM OTCA (). This suggests that the observed rescue effect of mitochondrial membrane potential is due to a compensatory increase in complex II activity rather than a rescue of the complex I defect.
Cell viability and cell growth are unaffected by parkin mutations
Cell viability and cell growth rates were very similar in fibroblasts from both controls and patients with parkin mutations (Cell viability - controls: 100% +/- 8.3%, mean +/- SD; parkin mutant patients 96% +/- 10.7%; cell growth — controls: 100% +/- 5.6%, parkin mutant patients: 95.4% +/- 14.3%). Furthermore, cell growth was not affected by pre-treatment of either controls or parkin-mutant patients cells with 10μM OTCA (controls: 102.3% +/- 9.6%, parkin mutant patients: 98% +/- 10.8%). Cell viability was increased in both patient and controls cells after pre-treatment with 10μM OTCA by approximately 8% (controls: 106.6% +/- 12.6%, parkin mutant patients: 104.3% +/- 18.8%), however this did not reach statistical significance (p > 0.05).