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Mitochondrial dysfunction in Parkinson disease: evidence in mutant PARK2 fibroblasts.

Zanellati MC, Monti V, Barzaghi C, Reale C, Nardocci N, Albanese A, Valente EM, Ghezzi D, Garavaglia B - Front Genet (2015)

Bottom Line: The mitochondrial network was comparable between mutant and control cells but, interestingly, a "chain-like" network was found only in mutant fibroblasts.The absence of mitochondrial fragmentation in mutant Parkin fibroblasts could results in accumulation of damaged mitochondria not targeted to mitophagy.This condition should increase the oxidative stress and lead to cellular dysfunction and death.

View Article: PubMed Central - PubMed

Affiliation: Unit of Molecular Neurogenetics - Pierfranco and Luisa Mariani Center for the Study of Mitochondrial Disorders in Children, Foundation of the Carlo Besta Neurological Institute IRCCS, Milan, Italy.

ABSTRACT
Mutations in PARK2, encoding Parkin, cause an autosomal recessive form of juvenile Parkinson Disease (JPD). The aim of the present study was to investigate the impact of PARK2 mutations on mitochondrial function and morphology in human skin fibroblasts. We analyzed cells obtained from four patients clinically characterized by JPD, harboring recessive mutations in PARK2. By quantitative PCR we found a reduction (<50%) of PARK2 transcript in all patients but one; however Western Blot analysis demonstrated the virtual absence of Parkin protein in all mutant fibroblasts. Respiration assays showed an increment of oxygen consumption, which was uncoupled to ATP cellular levels. This finding was probably due to presence of altered mitochondrial membrane potential (ΔΨm), confirmed by JC-1 analysis. The mitochondrial network was comparable between mutant and control cells but, interestingly, a "chain-like" network was found only in mutant fibroblasts. Dissipation of ΔΨm usually leads to mitochondrial fragmentation in healthy cells and eventually to mitophagy; however, this behavior was not observed in patients' fibroblasts. The absence of mitochondrial fragmentation in mutant Parkin fibroblasts could results in accumulation of damaged mitochondria not targeted to mitophagy. This condition should increase the oxidative stress and lead to cellular dysfunction and death. Our results suggest that PARK2 mutations cause mitochondrial impairment, in particular reduction in ATP cellular levels and alteration of ΔΨm, even in non-neuronal cells and confirm the hypothesis that Parkin holds a pivotal role in pro-fission events.

No MeSH data available.


Related in: MedlinePlus

Mitochondrial function. (A–D) The z-score values (dots) for basal oxygen consumption (OCR-B), maximal respiration rate (MRR), ATP amount, OCR-B minus OCR-O, are shown for control and mutant fibroblasts. Three independent experiments, each with 16 replicates, were performed. One-Way ANOVA followed by Tukey test was used (controls vs. mutant). (A) Mutant fibroblasts exhibited a significant increment of oxygen consumption. Pt1: p < 0.01 vs. Ct1,Ct2,Ct3,Ct4. Pt2: p < 0.05 vs. Ct1; p < 0.01 vs. Ct3. Pt3: p < 0.05 vs. Ct1; p < 0.01 vs. Ct3, Ct4. Pt4: p < 0.05 vs. Ct1, Ct4; p < 0.01 vs. Ct3. (B) With the exception of Pt3, mutant cells showed a higher MRR. Pt1, Pt2, Pt4: p < 0.01 vs. each Ct. (C) Analysis of ATP cellular levels showed a significant reduction in all mutant fibroblasts respect to controls. Pt1, Pt2, Pt4: p < 0.01 vs. each Ct; Pt3: p < 0.05 vs. Ct1, Ct2; p < 0.01 vs. Ct3, Ct4. (D) The difference between OCR-B and OCR-O has a trend toward reduction in mutant fibroblasts respect to controls. Pt1: p < 0.05 vs. Ct4; Pt2, Pt3, Pt4: p < 0.05 vs. Ct1, Ct2, Ct4.
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Figure 2: Mitochondrial function. (A–D) The z-score values (dots) for basal oxygen consumption (OCR-B), maximal respiration rate (MRR), ATP amount, OCR-B minus OCR-O, are shown for control and mutant fibroblasts. Three independent experiments, each with 16 replicates, were performed. One-Way ANOVA followed by Tukey test was used (controls vs. mutant). (A) Mutant fibroblasts exhibited a significant increment of oxygen consumption. Pt1: p < 0.01 vs. Ct1,Ct2,Ct3,Ct4. Pt2: p < 0.05 vs. Ct1; p < 0.01 vs. Ct3. Pt3: p < 0.05 vs. Ct1; p < 0.01 vs. Ct3, Ct4. Pt4: p < 0.05 vs. Ct1, Ct4; p < 0.01 vs. Ct3. (B) With the exception of Pt3, mutant cells showed a higher MRR. Pt1, Pt2, Pt4: p < 0.01 vs. each Ct. (C) Analysis of ATP cellular levels showed a significant reduction in all mutant fibroblasts respect to controls. Pt1, Pt2, Pt4: p < 0.01 vs. each Ct; Pt3: p < 0.05 vs. Ct1, Ct2; p < 0.01 vs. Ct3, Ct4. (D) The difference between OCR-B and OCR-O has a trend toward reduction in mutant fibroblasts respect to controls. Pt1: p < 0.05 vs. Ct4; Pt2, Pt3, Pt4: p < 0.05 vs. Ct1, Ct2, Ct4.

Mentions: In order to investigate mitochondrial bioenergetics status, we evaluated respiration and extracellular acidification by microscale oxyghraphy. We consistently observed a significant increment of oxygen consumption (basal OCR-B) in all mutant samples respect to controls (Figure 2A). Also the ratio OCR/ECAR was higher in mutant respect to controls whereas ECAR data was comparable between controls and mutant cells (Supplementary Figures S2A,B), indicating that metabolism of mutant cells did not shift toward glycolysis and that oxygen consumption alteration was directly linked to electron transfer chain activity. Similarly, MRR which is an index of electronic chain transport efficiency, was higher than controls in all mutant fibroblasts, except for Pt3 (Figure 2B). These findings indicated that mutant cells forced the activity of the respiratory chain in order to produce energy.


Mitochondrial dysfunction in Parkinson disease: evidence in mutant PARK2 fibroblasts.

Zanellati MC, Monti V, Barzaghi C, Reale C, Nardocci N, Albanese A, Valente EM, Ghezzi D, Garavaglia B - Front Genet (2015)

Mitochondrial function. (A–D) The z-score values (dots) for basal oxygen consumption (OCR-B), maximal respiration rate (MRR), ATP amount, OCR-B minus OCR-O, are shown for control and mutant fibroblasts. Three independent experiments, each with 16 replicates, were performed. One-Way ANOVA followed by Tukey test was used (controls vs. mutant). (A) Mutant fibroblasts exhibited a significant increment of oxygen consumption. Pt1: p < 0.01 vs. Ct1,Ct2,Ct3,Ct4. Pt2: p < 0.05 vs. Ct1; p < 0.01 vs. Ct3. Pt3: p < 0.05 vs. Ct1; p < 0.01 vs. Ct3, Ct4. Pt4: p < 0.05 vs. Ct1, Ct4; p < 0.01 vs. Ct3. (B) With the exception of Pt3, mutant cells showed a higher MRR. Pt1, Pt2, Pt4: p < 0.01 vs. each Ct. (C) Analysis of ATP cellular levels showed a significant reduction in all mutant fibroblasts respect to controls. Pt1, Pt2, Pt4: p < 0.01 vs. each Ct; Pt3: p < 0.05 vs. Ct1, Ct2; p < 0.01 vs. Ct3, Ct4. (D) The difference between OCR-B and OCR-O has a trend toward reduction in mutant fibroblasts respect to controls. Pt1: p < 0.05 vs. Ct4; Pt2, Pt3, Pt4: p < 0.05 vs. Ct1, Ct2, Ct4.
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Related In: Results  -  Collection

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Figure 2: Mitochondrial function. (A–D) The z-score values (dots) for basal oxygen consumption (OCR-B), maximal respiration rate (MRR), ATP amount, OCR-B minus OCR-O, are shown for control and mutant fibroblasts. Three independent experiments, each with 16 replicates, were performed. One-Way ANOVA followed by Tukey test was used (controls vs. mutant). (A) Mutant fibroblasts exhibited a significant increment of oxygen consumption. Pt1: p < 0.01 vs. Ct1,Ct2,Ct3,Ct4. Pt2: p < 0.05 vs. Ct1; p < 0.01 vs. Ct3. Pt3: p < 0.05 vs. Ct1; p < 0.01 vs. Ct3, Ct4. Pt4: p < 0.05 vs. Ct1, Ct4; p < 0.01 vs. Ct3. (B) With the exception of Pt3, mutant cells showed a higher MRR. Pt1, Pt2, Pt4: p < 0.01 vs. each Ct. (C) Analysis of ATP cellular levels showed a significant reduction in all mutant fibroblasts respect to controls. Pt1, Pt2, Pt4: p < 0.01 vs. each Ct; Pt3: p < 0.05 vs. Ct1, Ct2; p < 0.01 vs. Ct3, Ct4. (D) The difference between OCR-B and OCR-O has a trend toward reduction in mutant fibroblasts respect to controls. Pt1: p < 0.05 vs. Ct4; Pt2, Pt3, Pt4: p < 0.05 vs. Ct1, Ct2, Ct4.
Mentions: In order to investigate mitochondrial bioenergetics status, we evaluated respiration and extracellular acidification by microscale oxyghraphy. We consistently observed a significant increment of oxygen consumption (basal OCR-B) in all mutant samples respect to controls (Figure 2A). Also the ratio OCR/ECAR was higher in mutant respect to controls whereas ECAR data was comparable between controls and mutant cells (Supplementary Figures S2A,B), indicating that metabolism of mutant cells did not shift toward glycolysis and that oxygen consumption alteration was directly linked to electron transfer chain activity. Similarly, MRR which is an index of electronic chain transport efficiency, was higher than controls in all mutant fibroblasts, except for Pt3 (Figure 2B). These findings indicated that mutant cells forced the activity of the respiratory chain in order to produce energy.

Bottom Line: The mitochondrial network was comparable between mutant and control cells but, interestingly, a "chain-like" network was found only in mutant fibroblasts.The absence of mitochondrial fragmentation in mutant Parkin fibroblasts could results in accumulation of damaged mitochondria not targeted to mitophagy.This condition should increase the oxidative stress and lead to cellular dysfunction and death.

View Article: PubMed Central - PubMed

Affiliation: Unit of Molecular Neurogenetics - Pierfranco and Luisa Mariani Center for the Study of Mitochondrial Disorders in Children, Foundation of the Carlo Besta Neurological Institute IRCCS, Milan, Italy.

ABSTRACT
Mutations in PARK2, encoding Parkin, cause an autosomal recessive form of juvenile Parkinson Disease (JPD). The aim of the present study was to investigate the impact of PARK2 mutations on mitochondrial function and morphology in human skin fibroblasts. We analyzed cells obtained from four patients clinically characterized by JPD, harboring recessive mutations in PARK2. By quantitative PCR we found a reduction (<50%) of PARK2 transcript in all patients but one; however Western Blot analysis demonstrated the virtual absence of Parkin protein in all mutant fibroblasts. Respiration assays showed an increment of oxygen consumption, which was uncoupled to ATP cellular levels. This finding was probably due to presence of altered mitochondrial membrane potential (ΔΨm), confirmed by JC-1 analysis. The mitochondrial network was comparable between mutant and control cells but, interestingly, a "chain-like" network was found only in mutant fibroblasts. Dissipation of ΔΨm usually leads to mitochondrial fragmentation in healthy cells and eventually to mitophagy; however, this behavior was not observed in patients' fibroblasts. The absence of mitochondrial fragmentation in mutant Parkin fibroblasts could results in accumulation of damaged mitochondria not targeted to mitophagy. This condition should increase the oxidative stress and lead to cellular dysfunction and death. Our results suggest that PARK2 mutations cause mitochondrial impairment, in particular reduction in ATP cellular levels and alteration of ΔΨm, even in non-neuronal cells and confirm the hypothesis that Parkin holds a pivotal role in pro-fission events.

No MeSH data available.


Related in: MedlinePlus