Mutations in the PTEN-induced novel kinase 1 (Pink1) gene cause a recessive form of early onset parkinsonism, and all the evidence points to mitochondria as the prime locus of the damage. From its first identification, Pink1 was found to be important at keeping a healthy electrical balance across the mitochondrial membrane (see ARF related news story and Valente et al., 2004). Further studies in fruit flies showed that Pink1 knockout results in severe swelling and disorganization of mitochondria. These morphological effects were attributed to a lack of normal mitochondrial fission, as expression of the fission-promoting GTPase dynamin-related protein 1 (Drp1) compensated for the loss of Pink1 (see Park et al., 2006; Clark et al., 2006; and ARF related news story on Yang et al., 2008 and ARF related news story on Poole et al., 2008).

But data are accumulating in support of the controversial idea that the target for Pink1’s attack may lie elsewhere, that is, not in mitochondrial fission/fusion dynamics but at the heart of mitochondrial function itself, in energy production. The latest installment comes from Mark Cookson and colleagues at the National Institutes of Health in Bethesda, Maryland. In a paper published May 27 in PLoS ONE, the researchers show that knocking out Pink1 in a human neuroblastoma cell line adversely affects mitochondrial membrane potential and renders cells more sensitive to mitochondrial toxins. That finding was expected. But the morphological changes in the mitochondria, and the role of Drp1, were exactly opposite to that seen in flies. The lack of Pink1 produced smaller, more fragmented organelles, and promoting fission by expressing of Drp1 exacerbated cell toxicity. The morphological effects resolved when the cells were treated with the phosphatase inhibitor FK506, which blocked dephosphorylation and activation of Drp1; however, FK506 was unable to restore the membrane potential. The new results echo recent work from Bart De Strooper’s lab that suggested Pink1 inhibits the respiratory chain at Complex I, resulting in energy-starved dysfunctional synapses. This effect occurred in the absence of morphological changes in the organelles (see ARF related news story).

Cookson explained his group’s results in terms of primary and secondary effects of Pink1 loss. “The important thing we see is that although we can use FK506 to rescue some of the morphological effects, the functional effects were still there. This says that there is an order to things; there are early first events and there are later secondary events. Our interpretation of our data is that what Pink1 is really doing is maintaining mitochondrial function. And when you see subtle morphological effects, or grossly distended mitochondria, or nothing at all, that’s probably because those changes are secondary.”

To look at the effects of Pink1 on mitochondria function and morphology in living cells, first authors Anna Sandebring and Kelly Thomas made human dopaminergic M17 cell lines that overexpressed Pink1 or Pink1 shRNAs, and imaged mitochondria by a variety of techniques. Consistent with previous work, overexpression of Pink1 protected cells from the mitochondrial toxin rotenone, while the knockdown showed more cell death and mitochondrial fragmentation after treatment. Without Pink1, mitochondria had lower membrane potential and exhibited subtle morphological changes. The mitochondria appeared “a bit shorter and more fragmented,” Cookson told ARF. “It was not a huge effect, but it let us go into the mechanism.”

The scientists next examined the effect of Drp1. Partial knockdown of Drp1 prevented mitochondrial fragmentation in Pink1-minus cells, whereas overexpression of Drp1 made it worse. The results indicate that lack of Pink1 renders mitochondria sensitive to Drp1-induced fission. This was associated with a loss of Drp1 phosphorylation and increased GTPase activity. At the same time, cellular activity of the calcium-dependent Drp1 phosphatase calcineurin increased. Treating cells with the calcineurin inhibitor FK506 restored Drp1 phosphorylation levels, decreased the number of truncated or fragmented mitochondria, and restored the pattern of normal mitochondrial connection. However, FK506 did not cure the lower membrane potential.

From these results, the authors propose a model where loss of Pink1 has as its primary effect a loss of mitochondrial membrane potential. As a result, cells not only accumulate more reactive oxygen species, but they also fail to buffer calcium effectively. Increased calcium could activate calcineurin, leading to dephosphorylation of Drp1 and increased mitochondrial fission. In another species or cell type or developmental stage, the result on mitochondrial morphology might differ, Cookson said, depending on the regulation of Drp1 or other downstream effectors. In some cases, as in Pink1 knockout mice, there may be no morphological changes at all (Gautier et al., 2008). Nonetheless, Cookson says the primary deficit—a loss of mitochondrial health—is conserved.

Support for this idea comes from another recent paper from the lab of Charleen Chu at the University of Pittsburgh (Dagda et al., 2009). In that work, Pink1 knockdown in a different neuronal cell line, SH-SY5Y, also caused mitochondrial fragmentation. The paper suggests a role for mitochondria turnover via autophagy, which was also stimulated by Pink1 knockdown. The destruction of (presumably unhealthy) mitochondria was protective for the cells, and was enhanced by overexpression of the parkin protein, which has been implicated in autophagy (Narendra et al., 2008). The finding that parkin can compensate for Pink1 deficiency matches genetic studies in Drosophila, and Cookson reports that his group has similar, unpublished results in their cell lines showing a conservation of parkin function.

“If you look across all these studies, the observation that’s most clear is that the mitochondria are not at their full potential,” Cookson said. “It’s the details that are not clear.” The biggest detail Cookson is eager to settle is what Pink1 actually phosphorylates. “All of the evidence so far is that the kinase does not act directly on any of these fusion or fission proteins, but we haven’t looked at all of them. There is some evidence that it might phosphorylate parkin (see Kim et al., 2008), but I don’t know if that’s been reproduced.”—Pat McCaffrey


  1. The key difference between this study and the previous Drosophila studies is that a transformed neuroblastoma cell line is used here, whereas previous studies looked at postmitotic neurons and muscle in an in-vivo setting using Pink1-null mutant animals. It is known that mitochondria in proliferating cells and differentiated cells are very different, both in terms of morphology and function. This may account for the different conclusions from these studies. Future studies using postmitotic neurons, preferably dopaminergic neurons from Pink1 knockout animals, will be required to make fair comparisons between the studies.

  2. Accumulating evidence indicates that mitochondrial dysfunction is a key factor in the pathophysiology of Parkinson disease (PD). Inhibitors of complex I of the electron transport chain, such as MPTP and rotenone, can induce parkinsonism, and a decreased activity of complex I has indeed been detected in the substantia nigra of patients suffering from PD. Remarkably, the identification of genes associated with familial PD confirmed the crucial role of mitochondria for the integrity of dopaminergic neurons. One of the genes associated with autosomal-recessive PD encodes a bona fide mitochondrial protein, PINK1, a serine/threonine kinase that can protect cells against mitochondrial toxins.

    In Drosophila melanogaster, loss of PINK1 function causes apoptotic flight muscle degeneration and defective spermatogenesis, and mitochondrial pathology is the earliest manifestation of these phenotypes (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). PINK1 deficiency in flies can be rescued by increasing mitochondrial fission or decreasing fusion, leading to the conclusion that PINK1 promotes mitochondrial fission (Deng et al., 2008; Park et al., 2009; Poole et al., 2008; Yang et al., 2008). In contrast, when PINK1 is downregulated in cultured mammalian cells, an increase in mitochondrial fragmentation has been observed (Dagda et al., 2009; Exner et al., 2007; Weihofen et al., 2009).

    In the present study from Mark Cookson's group, this discrepancy was elegantly addressed. By using human neuroblastoma cells stably expressing wild-type PINK1, pathogenic PINK1 mutants or PINK1 shRNA, Sandebring and colleagues were able to demonstrate that increased expression of PINK1 can prevent mitochondrial fragmentation induced by rotenone treatment in a kinase-dependent fashion. In PINK1-deficient cells an increase in mitochondrial fragmentation was observed, and rotenone treatment added to that effect.

    Downregulation of Drp1, a GTPase promoting mitochondrial fission, significantly prevented mitochondrial fragmentation in PINK1-deficient cells. The activity of Drp1 is regulated by post-translational modifications; therefore, the authors analyzed whether in PINK1-deficient cells the phosphorylation status of Drp1 might be altered. They observed that loss of PINK1 function is associated with a decrease in phospho-Drp1 levels and an increase in Drp1 GTPase activity. Calcineurin is a phosphatase known to dephosphorylate Drp1 at S637; therefore, Sandebring and colleagues tested PINK1-deficient cells for expression levels and activity of calcineurin. They found that calcineurin activity was significantly increased in PINK1 knockdown cells, while expression levels were not affected. Interestingly, the calcineurin inhibitor FK506 ws able to rescue mitochondrial fragmentation caused by loss of PINK1 function.

    This study clearly shows that PINK1 silencing in human cells promotes mitochondrial fragmentation, which is dependent on Drp1 and involves calcineurin-mediated dephosphorylation of Drp1. How does this fit with what has been reported for PINK1-deficient flies? The most plausible explanation is that the phenotype observed in adult flies is influenced by compensatory effects. The accumulation of damaged mitochondria in PINK1-deficient flies might be a stimulus to activate fusion in an attempt to dilute these dysfunctional mitochondria (McBride, 2008). In tissues with high energy demand, such as flight muscles or spermatides, this strategy might not be efficient to remove damaged mitochondria. Thus, activation of mitochondrial fission might be beneficial, as it favors clearance of dysfunctional mitochondria by autophagy (Twig et al., 2008). All in all, the studies in mammalian cells and flies clearly demonstrate that PINK1 has an impact on mitochondrial function and integrity. It will now be important to unravel the underlying mechanism and to elaborate on the functional interaction between PINK1 and parkin.


    . Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature. 2006 Jun 29;441(7097):1162-6. PubMed.

    . Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission. J Biol Chem. 2009 May 15;284(20):13843-55. Epub 2009 Mar 10 PubMed.

    . The Parkinson's disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila. Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14503-8. PubMed.

    . Loss-of-function of human PINK1 results in mitochondrial pathology and can be rescued by parkin. J Neurosci. 2007 Nov 7;27(45):12413-8. PubMed.

    . Parkin mitochondria in the autophagosome. J Cell Biol. 2008 Dec 1;183(5):757-9. PubMed.

    . The PINK1-Parkin pathway is involved in the regulation of mitochondrial remodeling process. Biochem Biophys Res Commun. 2009 Jan 16;378(3):518-23. PubMed.

    . Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature. 2006 Jun 29;441(7097):1157-61. PubMed.

    . The PINK1/Parkin pathway regulates mitochondrial morphology. Proc Natl Acad Sci U S A. 2008 Feb 5;105(5):1638-43. PubMed.

    . Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J. 2008 Jan 23;27(2):433-46. PubMed.

    . Pink1 forms a multiprotein complex with Miro and Milton, linking Pink1 function to mitochondrial trafficking. Biochemistry. 2009 Mar 10;48(9):2045-52. PubMed.

    . Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin. Proc Natl Acad Sci U S A. 2006 Jul 11;103(28):10793-8. PubMed.

    . Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery. Proc Natl Acad Sci U S A. 2008 May 13;105(19):7070-5. PubMed.

  3. PINK1: Mitochondria bioenergetics dysfunction or deficient mitochondria dynamics? This question has raised some controversy in the Parkinson disease (PD) field in recent years. This matter is elegantly tackled in this paper from Sandebring and co-workers, where the authors attempt to address the underlying mechanism by which PINK1 influences mitochondrial morphology. In sum, they propose a mechanism where loss of PINK1 function causes decreased mitochondrial membrane potential (as has been previously reported by others), altering Ca2+ homeostasis, leading to the dephosphorylation of DRP1.

    These findings link the mitochondrial functional deficits with secondary mitochondria morphology alterations. Moreover, their findings are in complete agreement with our own work, recently published in EMBO Molecular Medicine [1], where we also claim that mitochondrial dysfunction is upstream of mitochondrial morphological defects observed in PINK1-deficient models. In our work, we also observed a decrease in mitochondrial membrane potential in two PINK1-deficient animal models (mouse and Drosophila).

    Furthermore, we have pinpointed this deficit to reduced Complex I activity. The reduced Complex I activity can be rescued by the human PINK1 wild-type form, but not by the familial PD mutations found in PINK1. Additionally, we also observe a synaptic deficit at the level of the neuromuscular junction of Drosophila larvae, which can be rescued with forward-fill of ATP, which strongly implicates an energy limitation.

    Sandebring et al. are careful to state that their proposal does not imply that PINK1 directly controls the mitochondrial membrane potential [1]. We, on the other hand, believe that the reduced activity of Complex I, which is known to cause reduced mitochondrial membrane potential, is due to the absence of PINK1. Whether a sub-unit of Complex I is a direct or indirect substrate for PINK1 is something that still needs to be addressed. At present, our efforts are devoted to identifying this promiscuous substrate that is leading to a mitochondrial bioenergetics dysfunction. Until we know this substrate, we are gratified that additional research groups corroborate our working hypothesis. Hopefully the “flurry” around PINK1, as also discussed by Anne Murphy in a Closeup of EMBO Molecular Medicine Journal [2], will strengthen the fact that these two aspects of mitochondrial morphology and bioenergetics are intimately associated. In future studies, one should take into consideration that mitochondria morphology alterations are downstream effects of a more complex deficiency occurring at the level of mitochondria function.


    . Parkinson's disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function. EMBO Mol Med. 2009 May;1(2):99-111. PubMed.

    . In a flurry of PINK, mitochondrial bioenergetics takes a leading role in Parkinson's disease. EMBO Mol Med. 2009 May;1(2):81-4. PubMed.

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News Citations

  1. Pink Mutations Link Parkinson’s Disease to Mitochondria
  2. Research Brief: A Swell Protein—Mitochondrial Fission Falls to Pink1, Again
  3. Pink Fission—Serving Up a Rationale for Parkinson Disease?
  4. That Touch of Pink1—Toning Mitochondrial Respiration

Paper Citations

  1. . Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature. 2006 Jun 29;441(7097):1157-61. PubMed.
  2. . Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature. 2006 Jun 29;441(7097):1162-6. PubMed.
  3. . Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery. Proc Natl Acad Sci U S A. 2008 May 13;105(19):7070-5. PubMed.
  4. . The PINK1/Parkin pathway regulates mitochondrial morphology. Proc Natl Acad Sci U S A. 2008 Feb 5;105(5):1638-43. PubMed.
  5. . Loss of PINK1 causes mitochondrial functional defects and increased sensitivity to oxidative stress. Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11364-9. PubMed.
  6. . Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission. J Biol Chem. 2009 May 15;284(20):13843-55. Epub 2009 Mar 10 PubMed.
  7. . Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol. 2008 Dec 1;183(5):795-803. Epub 2008 Nov 24 PubMed.
  8. . PINK1 controls mitochondrial localization of Parkin through direct phosphorylation. Biochem Biophys Res Commun. 2008 Dec 19;377(3):975-80. PubMed.

Further Reading

Primary Papers

  1. . Mitochondrial alterations in PINK1 deficient cells are influenced by calcineurin-dependent dephosphorylation of dynamin-related protein 1. PLoS One. 2009;4(5):e5701. PubMed.