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The principal PINK1 and Parkin cellular events triggered in response to dissipation of mitochondrial membrane potential occur in primary neurons.

Koyano F, Okatsu K, Ishigaki S, Fujioka Y, Kimura M, Sobue G, Tanaka K, Matsuda N - Genes Cells (2013)

Bottom Line: We found that dissipation of the mitochondrial membrane potential triggers phosphorylation of both PINK1 and Parkin and that, in response, Parkin translocates to depolarized mitochondria.Furthermore, Parkin's E3 activity is re-established concomitant with ubiquitin-ester formation at Cys431 of Parkin.As a result, mitochondrial substrates in neurons become ubiquitylated.

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Affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.

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Disease-relevant Parkin mutations impair mitochondrial localization and E3 activity after CCCP treatment. (A) The subcellular localization of GFP-Parkin with pathogenic mutations in the isolated neurons from PARKIN knockout (PARKIN−/−) mice. Primary neurons were infected with lentivirus encoding GFP-Parkin containing various disease-relevant mutations and then treated with CCCP (30 μm) for 3 h, followed by immunocytochemistry, as in Fig. 2A. (B) The number of neurons with GFP-Parkin-positive mitochondria was counted. Error bars represent the mean ± SD values of two experiments. Statistical significance was calculated using analysis of variance with a Student's t-test. (C) The E3 activity of Parkin with disease-relevant Parkin mutations. PARKIN−/− primary neurons expressing pathogenic GFP-Parkin were treated with CCCP for 3 h and subjected to immunoblotting with an anti-Parkin antibody.
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fig03: Disease-relevant Parkin mutations impair mitochondrial localization and E3 activity after CCCP treatment. (A) The subcellular localization of GFP-Parkin with pathogenic mutations in the isolated neurons from PARKIN knockout (PARKIN−/−) mice. Primary neurons were infected with lentivirus encoding GFP-Parkin containing various disease-relevant mutations and then treated with CCCP (30 μm) for 3 h, followed by immunocytochemistry, as in Fig. 2A. (B) The number of neurons with GFP-Parkin-positive mitochondria was counted. Error bars represent the mean ± SD values of two experiments. Statistical significance was calculated using analysis of variance with a Student's t-test. (C) The E3 activity of Parkin with disease-relevant Parkin mutations. PARKIN−/− primary neurons expressing pathogenic GFP-Parkin were treated with CCCP for 3 h and subjected to immunoblotting with an anti-Parkin antibody.

Mentions: To further verify that the events shown in Fig. 2 are aetiologically important, we selected six pathogenic mutants of Parkin (K211N, T240R, R275W, C352G, T415N and G430D) and examined their subcellular localization and E3 activity. To eliminate the effect of endogenous Parkin, we used primary neurons derived from PARKIN−/− mice in these experiments. The six GFP-Parkin mutants were serially introduced into PARKIN−/− primary neurons using a lentivirus and assayed for their subcellular localization after CCCP treatment. Parkin mitochondrial localization was compromised by the K211N (mutation in RING0 domain), T240R (in RING1 domain), C352G (in IBR domain), T415N and G430D (both in RING2 domain) mutations (Fig. 3A). The defects seen with the K211N, T240R, C352G and G430D mutants (Fig. 3B), in contrast to T415N (P > 0.01), were statistically significant (P < 0.01). The R275W mutation had no effect on mitochondrial localization after CCCP treatment. The E3 activity of the mutants was also assessed. The K211N, T240R, C352G, T415N and G430D mutations exhibited deficient autoubiquitylation activity in PARKIN−/− primary neurons (Fig. 3C). The R275W mutant had weak but reproducible autoubiquitylation activity after CCCP treatment. Because this mutant showed partial mitochondrial localization after CCCP treatment even in HeLa cells (Okatsu et al. 31; Lazarou et al. 20), it is not surprising that the R275W mutant localizes to neuronal depolarized mitochondria and possesses weak E3 activity. Unexpectedly, the R275W mutant also localized to mitochondria even in the absence of CCCP treatment. Although the significance of R275W localization to healthy mitochondria is unknown, we propose that the R275W mutation maintains Parkin in an inactive state (as suggested by Fig. 3C) because functional, phosphorylated PINK1 has not been reported in normal mitochondria. In most of the pathogenic Parkin mutants, translocation to damaged mitochondria and conversion to the active form were compromised after a decrease in ΔΨm (Fig. 3), suggesting the aetiological importance of these events in neurons.


The principal PINK1 and Parkin cellular events triggered in response to dissipation of mitochondrial membrane potential occur in primary neurons.

Koyano F, Okatsu K, Ishigaki S, Fujioka Y, Kimura M, Sobue G, Tanaka K, Matsuda N - Genes Cells (2013)

Disease-relevant Parkin mutations impair mitochondrial localization and E3 activity after CCCP treatment. (A) The subcellular localization of GFP-Parkin with pathogenic mutations in the isolated neurons from PARKIN knockout (PARKIN−/−) mice. Primary neurons were infected with lentivirus encoding GFP-Parkin containing various disease-relevant mutations and then treated with CCCP (30 μm) for 3 h, followed by immunocytochemistry, as in Fig. 2A. (B) The number of neurons with GFP-Parkin-positive mitochondria was counted. Error bars represent the mean ± SD values of two experiments. Statistical significance was calculated using analysis of variance with a Student's t-test. (C) The E3 activity of Parkin with disease-relevant Parkin mutations. PARKIN−/− primary neurons expressing pathogenic GFP-Parkin were treated with CCCP for 3 h and subjected to immunoblotting with an anti-Parkin antibody.
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fig03: Disease-relevant Parkin mutations impair mitochondrial localization and E3 activity after CCCP treatment. (A) The subcellular localization of GFP-Parkin with pathogenic mutations in the isolated neurons from PARKIN knockout (PARKIN−/−) mice. Primary neurons were infected with lentivirus encoding GFP-Parkin containing various disease-relevant mutations and then treated with CCCP (30 μm) for 3 h, followed by immunocytochemistry, as in Fig. 2A. (B) The number of neurons with GFP-Parkin-positive mitochondria was counted. Error bars represent the mean ± SD values of two experiments. Statistical significance was calculated using analysis of variance with a Student's t-test. (C) The E3 activity of Parkin with disease-relevant Parkin mutations. PARKIN−/− primary neurons expressing pathogenic GFP-Parkin were treated with CCCP for 3 h and subjected to immunoblotting with an anti-Parkin antibody.
Mentions: To further verify that the events shown in Fig. 2 are aetiologically important, we selected six pathogenic mutants of Parkin (K211N, T240R, R275W, C352G, T415N and G430D) and examined their subcellular localization and E3 activity. To eliminate the effect of endogenous Parkin, we used primary neurons derived from PARKIN−/− mice in these experiments. The six GFP-Parkin mutants were serially introduced into PARKIN−/− primary neurons using a lentivirus and assayed for their subcellular localization after CCCP treatment. Parkin mitochondrial localization was compromised by the K211N (mutation in RING0 domain), T240R (in RING1 domain), C352G (in IBR domain), T415N and G430D (both in RING2 domain) mutations (Fig. 3A). The defects seen with the K211N, T240R, C352G and G430D mutants (Fig. 3B), in contrast to T415N (P > 0.01), were statistically significant (P < 0.01). The R275W mutation had no effect on mitochondrial localization after CCCP treatment. The E3 activity of the mutants was also assessed. The K211N, T240R, C352G, T415N and G430D mutations exhibited deficient autoubiquitylation activity in PARKIN−/− primary neurons (Fig. 3C). The R275W mutant had weak but reproducible autoubiquitylation activity after CCCP treatment. Because this mutant showed partial mitochondrial localization after CCCP treatment even in HeLa cells (Okatsu et al. 31; Lazarou et al. 20), it is not surprising that the R275W mutant localizes to neuronal depolarized mitochondria and possesses weak E3 activity. Unexpectedly, the R275W mutant also localized to mitochondria even in the absence of CCCP treatment. Although the significance of R275W localization to healthy mitochondria is unknown, we propose that the R275W mutation maintains Parkin in an inactive state (as suggested by Fig. 3C) because functional, phosphorylated PINK1 has not been reported in normal mitochondria. In most of the pathogenic Parkin mutants, translocation to damaged mitochondria and conversion to the active form were compromised after a decrease in ΔΨm (Fig. 3), suggesting the aetiological importance of these events in neurons.

Bottom Line: We found that dissipation of the mitochondrial membrane potential triggers phosphorylation of both PINK1 and Parkin and that, in response, Parkin translocates to depolarized mitochondria.Furthermore, Parkin's E3 activity is re-established concomitant with ubiquitin-ester formation at Cys431 of Parkin.As a result, mitochondrial substrates in neurons become ubiquitylated.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.

Show MeSH
Related in: MedlinePlus