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PINK1-associated Parkinson's disease is caused by neuronal vulnerability to calcium-induced cell death.

Gandhi S, Wood-Kaczmar A, Yao Z, Plun-Favreau H, Deas E, Klupsch K, Downward J, Latchman DS, Tabrizi SJ, Wood NW, Duchen MR, Abramov AY - Mol. Cell (2009)

Bottom Line: PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload.ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration.Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neuroscience, Institute of Neurology, Queen Square, London, UK.

ABSTRACT
Mutations in PINK1 cause autosomal recessive Parkinson's disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na(+)/Ca(2+) exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload. We show that calcium overload stimulates reactive oxygen species (ROS) production via NADPH oxidase. ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration. We demonstrate that impaired respiration may be restored by provision of mitochondrial complex I and II substrates. Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells. Our findings propose a mechanism by which PINK1 dysfunction renders neurons vulnerable to cell death.

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Physiological Calcium Stimuli Induce Mitochondrial Depolarization in PINK1 KD Cells(A–C) Mouse (Ai and Aii) and human (Bi–Biii) neurons were loaded with fura-2 am and Rh123. KCl (50 mM) produced a rise in [Ca2+]c in mouse wild-type neurons (Ai) and human control neurons (Bi). In PINK1 KO mouse neurons (Aii) and in PINK1 KD human neurons (Bii), the [Ca2+]c was associated with an increase in Rh123 fluorescence and Δψm depolarization. Preincubation of human PINK1 KD neurons with 0.5 μM CsA prevented the Δψm depolarization of cells, but not the [Ca2+]c signal (C).(D and E) Human neuroblastoma cells were loaded with fluo-4 (green) and TMRM (red). ATP (100 μM) induced a rise in [Ca2+]c and thus an increase in fluo-4 fluorescence. In PINK1 KD cells the ATP-induced [Ca2+]c signal was associated with a decrease in TMRM fluorescence and Δψm depolarization. Error bars represent mean ± SEM.
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fig3: Physiological Calcium Stimuli Induce Mitochondrial Depolarization in PINK1 KD Cells(A–C) Mouse (Ai and Aii) and human (Bi–Biii) neurons were loaded with fura-2 am and Rh123. KCl (50 mM) produced a rise in [Ca2+]c in mouse wild-type neurons (Ai) and human control neurons (Bi). In PINK1 KO mouse neurons (Aii) and in PINK1 KD human neurons (Bii), the [Ca2+]c was associated with an increase in Rh123 fluorescence and Δψm depolarization. Preincubation of human PINK1 KD neurons with 0.5 μM CsA prevented the Δψm depolarization of cells, but not the [Ca2+]c signal (C).(D and E) Human neuroblastoma cells were loaded with fluo-4 (green) and TMRM (red). ATP (100 μM) induced a rise in [Ca2+]c and thus an increase in fluo-4 fluorescence. In PINK1 KD cells the ATP-induced [Ca2+]c signal was associated with a decrease in TMRM fluorescence and Δψm depolarization. Error bars represent mean ± SEM.

Mentions: Mitochondria play a major role in maintaining neuronal calcium homeostasis (Szabadkai et al., 2006). In order to examine mitochondrial calcium handling in the PINK1 KD/KO cells, we measured fura-2 and Rh123 fluorescence (dequench mode) simultaneously as indicators of [Ca2+]c and Δψm. Application of 50 mM KCl depolarizes the plasma membrane in neurons, induces the opening of potential-sensitive calcium channels, causing a rise in [Ca2+]c. Application of 50 mM KCl to WT mouse neurons produced a [Ca2+]c signal, with no significant mitochondrial depolarization as characterized before (Keelan et al., 1999) (Figure 3Ai). KCl (50 mM) application to PINK1 KO neurons induced a much higher [Ca2+]c signal (signal increase: control 0.52 ± 0.001, n = 134, compared to 1.12 ± 0.1 in PINK1 KD, n = 187; p < 0.001; Figure 3Aii), which was associated with a major loss of Δψm (the Rh123 signal rose by 68.9% ± 5.7%, n = 187) (Figure 3Aii). In human neurons, KCl induced a small rise in the [Ca2+]c in control neurons (Figure 3Bi) and a much higher [Ca2+]c in PINK1 KD neurons (0.36 ± 0.1, n = 133 in control compared to 0.76 ± 0.41, n = 102; Figure 3Bii). The higher [Ca2+]c in PINK1 KD neurons was associated with profound lowering of Δψm (Rh123 signal increased by 94% ± 6.6%). The mitochondrial depolarization was prevented by preincubation with the mPTP inhibitor cyclosporin A (CsA) (0.5 μM; Figure 3C).


PINK1-associated Parkinson's disease is caused by neuronal vulnerability to calcium-induced cell death.

Gandhi S, Wood-Kaczmar A, Yao Z, Plun-Favreau H, Deas E, Klupsch K, Downward J, Latchman DS, Tabrizi SJ, Wood NW, Duchen MR, Abramov AY - Mol. Cell (2009)

Physiological Calcium Stimuli Induce Mitochondrial Depolarization in PINK1 KD Cells(A–C) Mouse (Ai and Aii) and human (Bi–Biii) neurons were loaded with fura-2 am and Rh123. KCl (50 mM) produced a rise in [Ca2+]c in mouse wild-type neurons (Ai) and human control neurons (Bi). In PINK1 KO mouse neurons (Aii) and in PINK1 KD human neurons (Bii), the [Ca2+]c was associated with an increase in Rh123 fluorescence and Δψm depolarization. Preincubation of human PINK1 KD neurons with 0.5 μM CsA prevented the Δψm depolarization of cells, but not the [Ca2+]c signal (C).(D and E) Human neuroblastoma cells were loaded with fluo-4 (green) and TMRM (red). ATP (100 μM) induced a rise in [Ca2+]c and thus an increase in fluo-4 fluorescence. In PINK1 KD cells the ATP-induced [Ca2+]c signal was associated with a decrease in TMRM fluorescence and Δψm depolarization. Error bars represent mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724101&req=5

fig3: Physiological Calcium Stimuli Induce Mitochondrial Depolarization in PINK1 KD Cells(A–C) Mouse (Ai and Aii) and human (Bi–Biii) neurons were loaded with fura-2 am and Rh123. KCl (50 mM) produced a rise in [Ca2+]c in mouse wild-type neurons (Ai) and human control neurons (Bi). In PINK1 KO mouse neurons (Aii) and in PINK1 KD human neurons (Bii), the [Ca2+]c was associated with an increase in Rh123 fluorescence and Δψm depolarization. Preincubation of human PINK1 KD neurons with 0.5 μM CsA prevented the Δψm depolarization of cells, but not the [Ca2+]c signal (C).(D and E) Human neuroblastoma cells were loaded with fluo-4 (green) and TMRM (red). ATP (100 μM) induced a rise in [Ca2+]c and thus an increase in fluo-4 fluorescence. In PINK1 KD cells the ATP-induced [Ca2+]c signal was associated with a decrease in TMRM fluorescence and Δψm depolarization. Error bars represent mean ± SEM.
Mentions: Mitochondria play a major role in maintaining neuronal calcium homeostasis (Szabadkai et al., 2006). In order to examine mitochondrial calcium handling in the PINK1 KD/KO cells, we measured fura-2 and Rh123 fluorescence (dequench mode) simultaneously as indicators of [Ca2+]c and Δψm. Application of 50 mM KCl depolarizes the plasma membrane in neurons, induces the opening of potential-sensitive calcium channels, causing a rise in [Ca2+]c. Application of 50 mM KCl to WT mouse neurons produced a [Ca2+]c signal, with no significant mitochondrial depolarization as characterized before (Keelan et al., 1999) (Figure 3Ai). KCl (50 mM) application to PINK1 KO neurons induced a much higher [Ca2+]c signal (signal increase: control 0.52 ± 0.001, n = 134, compared to 1.12 ± 0.1 in PINK1 KD, n = 187; p < 0.001; Figure 3Aii), which was associated with a major loss of Δψm (the Rh123 signal rose by 68.9% ± 5.7%, n = 187) (Figure 3Aii). In human neurons, KCl induced a small rise in the [Ca2+]c in control neurons (Figure 3Bi) and a much higher [Ca2+]c in PINK1 KD neurons (0.36 ± 0.1, n = 133 in control compared to 0.76 ± 0.41, n = 102; Figure 3Bii). The higher [Ca2+]c in PINK1 KD neurons was associated with profound lowering of Δψm (Rh123 signal increased by 94% ± 6.6%). The mitochondrial depolarization was prevented by preincubation with the mPTP inhibitor cyclosporin A (CsA) (0.5 μM; Figure 3C).

Bottom Line: PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload.ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration.Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neuroscience, Institute of Neurology, Queen Square, London, UK.

ABSTRACT
Mutations in PINK1 cause autosomal recessive Parkinson's disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na(+)/Ca(2+) exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload. We show that calcium overload stimulates reactive oxygen species (ROS) production via NADPH oxidase. ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration. We demonstrate that impaired respiration may be restored by provision of mitochondrial complex I and II substrates. Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells. Our findings propose a mechanism by which PINK1 dysfunction renders neurons vulnerable to cell death.

Show MeSH
Related in: MedlinePlus