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.

View all comments by Bingwei Lu

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...  Read more

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.

References:
Clark, I.E., Dodson, M.W., Jiang, C., Cao, J.H., Huh, J.R., Seol, J.H., Yoo, S.J., Hay, B.A. and Guo, M. (2006) Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature, 441, 1162-1166. Abstract

Dagda, R.K., Cherra, S.J., 3rd, Kulich, S.M., Tandon, A., Park, D. and Chu, C.T. (2009) Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission. J Biol Chem, 284, 13843-13855. Abstract

Deng, H., Dodson, M.W., Huang, H. and Guo, M. (2008) The Parkinson's disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila. Proc Natl Acad Sci U S A, 105, 14503-14508. Abstract

Exner, N., Treske, B., Paquet, D., Holmström, K., Schiesling, C., Gispert, S., Carballo-Carbajal, I., Berg, D., Hoepken, H.-H., Gasser, T., Krüger, R., Winklhofer, K.F., Vogel, F., Reichert, A., Auburger, G., Kahle, P.J., Schmid, B. and Haass, C. (2007) Loss-of-function of human PINK1 results in mitochondrial pathology and can be rescued by parkin. J Neurosci, 27, 12413-12418. Abstract

McBride, H.M. (2008) Parkin mitochondria in the autophagosome. J Cell Biol, 183, 757-759. Abstract

Park, J., Lee, G. and Chung, J. (2009) The PINK1-Parkin pathway is involved in the regulation of mitochondrial remodeling process. Biochem Biophys Res Commun, 378, 518-523. Abstract

Park, J., Lee, S.B., Lee, S., Kim, Y., Song, S., Kim, S., Bae, E., Kim, J., Shong, M., Kim, J.M. and Chung, J. (2006) Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature, 441, 1157-1161. Abstract

Poole, A.C., Thomas, R.E., Andrews, L.A., McBride, H.M., Whitworth, A.J. and Pallanck, L.J. (2008) The PINK1/Parkin pathway regulates mitochondrial morphology. Proc Natl Acad Sci U S A, 105, 1638-1643. Abstract

Twig, G., Elorza, A., Molina, A.J., Mohamed, H., Wikstrom, J.D., Walzer, G., Stiles, L., Haigh, S.E., Katz, S., Las, G., Alroy, J., Wu, M., Py, B.F., Yuan, J., Deeney, J.T., Corkey, B.E. and Shirihai, O.S. (2008) Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. Embo J, 27, 433-446. Abstract

Weihofen, A., Thomas, K.J., Ostaszewski, B.L., Cookson, M.R. and Selkoe, D.J. (2009) Pink1 forms a multiprotein complex with Miro and Milton, linking Pink1 function to mitochondrial trafficking. Biochemistry, 48, 2045-2052. Abstract

Yang, Y., Gehrke, S., Imai, Y., Huang, Z., Ouyang, Y., Wang, J.W., Yang, L., Beal, M.F., Vogel, H. and Lu, B. (2006) 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, 103, 10793-10798. Abstract

Yang, Y., Ouyang, Y., Yang, L., Beal, M.F., McQuibban, A., Vogel, H. and Lu, B. (2008) Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery. Proc Natl Acad Sci U S A, 105, 7070-7075. Abstract

View all comments by Konstanze Winklhofer

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...  Read more

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.

References:
1. V.A. Morais, P. Verstreken, A. Roethig, J. Smet, A. Snellinx, M. Vanbrabant, D. Haddad, C. Frezza, W. Mandemakers, D. Vogt-Weisenhorn, R. Van Coster, W. Wurst, L. Scorrano, B. De Strooper (2009) Parkinson's disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function. EMBO Molecular Medicine; 1(2): 99-111.

2. A. Murphy (2009) In a flurry of PINK, mitochondrial bioenergetics takes a leading role in Parkinson's disease. EMBO Molecular Medicine; 1(2): 81-84.

View all comments by Vanessa Morais

The cloning of the gene for the PARK6 locus by Enza-Maria Valente and her colleagues now gives us a triumvirate of recessive genes that cause parkinsonism in humans; parkin, DJ-1 and now Pink1. Logically, the identification of three recessive mutations with similar phenotypes suggests that either 1) these three genes now delineate a single pathogenic pathway or 2) they point to different pathogenic processes that happen to all cause loss of a small group of neurons in the substantia nigra.

PINK1 looks very much like a serine/threonine-directed protein kinase, and thus has no immediate connection to the E3-ligase activity of parkin or the varied putative activities of DJ-1. This suggests the involvement of diverse cellular pathways. However, there are one or two intersecting observations that may indicate some similarities among these different gene products. The first is that Pink1 localizes to mitochondria. DJ-1 can localize to mitochondria under some circumstances, and a fraction of parkin is also found in this organelle. The proportion of parkin that localizes to...  Read more

The cloning of the gene for the PARK6 locus by Enza-Maria Valente and her colleagues now gives us a triumvirate of recessive genes that cause parkinsonism in humans; parkin, DJ-1 and now Pink1. Logically, the identification of three recessive mutations with similar phenotypes suggests that either 1) these three genes now delineate a single pathogenic pathway or 2) they point to different pathogenic processes that happen to all cause loss of a small group of neurons in the substantia nigra.

PINK1 looks very much like a serine/threonine-directed protein kinase, and thus has no immediate connection to the E3-ligase activity of parkin or the varied putative activities of DJ-1. This suggests the involvement of diverse cellular pathways. However, there are one or two intersecting observations that may indicate some similarities among these different gene products. The first is that Pink1 localizes to mitochondria. DJ-1 can localize to mitochondria under some circumstances, and a fraction of parkin is also found in this organelle. The proportion of parkin that localizes to mitochondria is small, but experiments in mice and flies suggest that there are mitochondrial effects of parkin knockout. The other thing that links these three proteins is that all three protect cells against “stress” in a very broad sense. Thus, parkin protects against proteasome inhibition and mitochondrial stress, DJ-1 against oxidative events (which may be mitochondrial in nature) and proteasome inhibition, and Pink1 protects against proteasomal dysfunction and mitochondrial damage. Which leaves us with a number of important questions to answer. The mechanism by which Pink1 protects cells against mitochondrial damage secondary to proteasome inhibition is unclear and Valente et al. evoke a mitochondrial substrate. So what are the kinase substrates of Pink1? And is Pink1 somehow intertwined with parkin and DJ-1; or is the concept of one pathogenic cascade a red (or pink) herring?

View all comments by Mark Cookson

The two exciting reports in ScienceExpress of two discoveries, one, the mutant Pink1 gene at the root of PARK6-linked autosomal recessive Parkinson disease; and two, the functional inactivation of parkin's ubiquitin ligase activity by S-nitrosylation, provide strong support for an integrated picture of Parkinson's disease. The characterizations of Pink1 localization (and thus, likely, function) in mitochondria and parkin's inactivation as a result of excess oxidative stress cement two cornerstones of PD pathogenesis, mitochondrial impairment and sustained oxidative stress. They also highlight the relevance of wild-type parkin in the development of sporadic, late-onset PD, given its role in regulating steady-state levels of both mitochondrial enzymes and antioxidant proteins in parkin-deficient mouse brain (see Palacino et al., 2004 in ARF related news story).

View all comments by Michael Schlossmacher

We appreciate the comments of Dr. Pallanck on our work, but would like to add some additional information that will allow the reader to put this criticism in perspective. First, it should be noted that no molecular mechanism has been provided for the putative role of Pink1 in mitochondrial fission, and that our work provides an experimentally well-supported alternative mechanism to explain the available observations. We refer to our paper for further discussion.

More importantly, some additional background on the experiments of Choi et al., as cited by Dr. Pallanck, will shed more light on the interpretation of these data. The animal toxin models that use mitochondrial Complex I inhibitors, such as MPTP and rotenone, to induce PD-like symptoms are widely used to study PD. Studies performed by researchers led by Zhengui Xia and Richard Palmiter have reported that upon deletion of an assembly factor of Complex I, Ndufs4, dopaminergic neurons remain sensitive to well-established Complex I mitochondrial inhibitors, arguing that dopaminergic neuron loss is not due to Complex I...  Read more

We appreciate the comments of Dr. Pallanck on our work, but would like to add some additional information that will allow the reader to put this criticism in perspective. First, it should be noted that no molecular mechanism has been provided for the putative role of Pink1 in mitochondrial fission, and that our work provides an experimentally well-supported alternative mechanism to explain the available observations. We refer to our paper for further discussion.

More importantly, some additional background on the experiments of Choi et al., as cited by Dr. Pallanck, will shed more light on the interpretation of these data. The animal toxin models that use mitochondrial Complex I inhibitors, such as MPTP and rotenone, to induce PD-like symptoms are widely used to study PD. Studies performed by researchers led by Zhengui Xia and Richard Palmiter have reported that upon deletion of an assembly factor of Complex I, Ndufs4, dopaminergic neurons remain sensitive to well-established Complex I mitochondrial inhibitors, arguing that dopaminergic neuron loss is not due to Complex I inhibition (Choi et al., 2008). However, the original report by Palmiter and colleagues (Kruse et al., 2008) shows clearly that tissue from Ndufs4-/- mice retains approximately 50 percent of respiration driven by Complex I substrates. This is in complete accordance with the 50 percent reduction of assembled Complex I retrieved by blue native PAGE in the tissue from these mice. Of note, rotenone completely inhibits the residual respiration of Ndufs4-/- mitochondria, indicating that this Complex I inhibitor is still working in this genetic background. The complete lack of Complex I activity in submitochondrial particles is likely a consequence of the harsh procedure (e.g., sonication) required for their preparation and in no way should be taken as a proof of lack of Complex I in these mitochondria. As correctly pointed out by the authors of the Choi et al. study, “…. Thus, one could argue that partially assembled complex I, although lacking complex I activity and the ability to generate NAD+, could still transfer electrons. This may explain the toxicity of rotenone and MPP+ in Ndufs4-/-; neurons.”

In conclusion, these findings do not indicate that the residual and putatively unstable Complex I that is formed in these mice is not capable of being affected by these specific inhibitors. On the contrary, rotenone does inhibit respiration and therefore electron transfer at Complex I even in Ndufs4-/- mitochondria. Therefore, it is likely that rotenone and MPTP do not have Complex I independent effects on mitochondria, and that their effect on dopaminergic neuron viability is solely related to Complex I inhibition.

View all comments by Bart De Strooper
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