Mitochondria take the stage in three recent papers—two that flesh out how several Parkinson disease-related proteins regulate degradation of these organelles, and one that finds loss of synaptic mitochondria as an early phenotype in a new Drosophila model for Alzheimer disease. Reporting in the January issue of PLoS Biology, researchers led by Richard Youle, National Institutes of Health, Bethesda, Maryland, describe how Pink1 and parkin work together to help cells deal with malfunctioning mitochondria. Another study—led by Wolfdieter Springer, University of Tübingen, Germany, and published online January 24 in Nature Cell Biology—unravels downstream events of the same pathway, showing how Pink1 and parkin link mitochondrial dysfunction with ubiquitination and eventual autophagy. And last but not least, a research team led by Fu-De Huang, Chinese Academy of Sciences, Shanghai, reports in the January 27 Journal of Neuroscience that Aβ-overexpressing flies lose presynaptic mitochondria before developing obvious problems with synaptic transmission. Using different model systems, the new studies strengthen the case that mitochondrial dysfunction contributes to neurodegenerative disease pathogenesis.

Previous studies have fingered mitochondria as possibly contributing to PD (see ARF related news story), and mitochondrial dynamics are thought to be disrupted by mutations in Pink1 (a mitochondrial kinase) or parkin (an E3 ubiquitin ligase). Loss-of-function mutations in either can cause PD, and the two proteins interact genetically (Clark et al., 2006), with Pink1 acting upstream of parkin (Exner et al., 2007; Park et al., 2006). Furthermore, parkin translocates to damaged mitochondria, triggering their autophagy (aka mitophagy) (Narendra et al., 2008).

As reported in PLoS Biology, Youle, first author Derek Narendra, and colleagues did a series of biochemistry experiments that show how Pink1 helps parkin sense flagging mitochondria in the first place. In their model—derived from experiments using a host of cultured cell types, both neuronal and non-neuronal—cells keep Pink1 at low levels in healthy mitochondria but selectively stabilize the protein on distressed mitochondria. The extra Pink1 signals to parkin, which tags the mitochondria for destruction by autophagy. The researchers went on to show that pathogenic mutations in Pink1 or parkin disrupt this pathway, suggesting that PD may be caused by a failure to clear damaged mitochondria, leading to neuronal death.

In the Nature Cell Biology study, Springer, first author Sven Giesler, and colleagues examined the same Pink1/parkin pathway but focused further downstream—namely, on how parkin’s recruitment to stressed mitochondria promotes mitophagy. The researchers treated neuronal and non-neuronal cultured cells with a compound (CCCP) that induces mitochondrial depolarization, and used immunostaining to follow Pink1 and parkin in the context of ensuing mitochondrial demise. They found that mitophagy requires functional parkin, as cells transfected with pathogenic parkin mutants failed to clear damaged mitochondria. Blocking Pink1 with short interfering RNA and then counteracting the knockdown by transfecting in wild-type protein, the researchers showed that Pink1 is required to recruit parkin to dysfunctional mitochondria. In studies with various Pink1 mutants, they further determined that Pink1 needs proper kinase activity and mitochondrial targeting sequences to direct itself and parkin to damaged mitochondria. Once there, parkin promotes formation of two poly-ubiquitin chains. The researchers identified voltage-dependent anion channel 1 (VDAC1) as a potential target for this ubiquitination, and showed that downstream steps leading from ubiquitination to mitophagy require the adaptor protein p62/SQSTM1. Like Youle and colleagues, Springer’s team shows that PD-associated parkin mutations stymie specific steps within the cascade linking mitochondrial damage, ubiquitination, and autophagy.

“The authors did a marvelous job in providing biochemical and cell biological evidence linking the Pink1/parkin pathway to mitochondrial autophagy,” wrote Bingwei Lu of Stanford University, Palo Alto, California, in an e-mail to ARF. “What remains to be determined, though, is the in vivo relevance of the findings to dopaminergic neuron maintenance and survival.” (See full comment below.) Toward this end, the German researchers have begun studying neurons from parkin knockout mice and neuronal cells derived from reprogrammed fibroblasts of PD patients, Springer told ARF.

David Park of the University of Ottawa, Canada, agrees that the relevance of the current findings to PD pathogenesis remains uncertain. Nevertheless, “It’s an exciting paper,” he said. “I think there will be more stories coming out in support of this theme.” Park suspects that parkin-mediated mitophagy could turn out to be a general mechanism of mitochondrial quality control with potential implications for normal aging and for other neurodegenerative diseases besides PD.

Abnormal mitochondrial dynamics have also been linked to AD (Wang et al., 2009 and ARF related news story), and the Journal of Neuroscience paper by Huang’s team supports this. First author Xiao-Liang Zhao and colleagues created a new AD fly model by expressing mutant human Aβ in the giant fiber neurons that control flight, and tracking axonal and synaptic changes in the flies using electrophysiology and microscopy studies.

Besides decreased mobility and faster demise, the Aβ-expressing flies developed a host of abnormalities, most notably loss of presynaptic mitochondria, which preceded appearance of synaptic transmission problems. “It’s striking that mitochondrial depletion from axons and presynaptic terminals was the earliest detected phenotype. That’s what I tried to find in in vivo models,” said Xiongwei Zhu of Case Western Reserve University in Cleveland, Ohio, who was not involved in this work. In a recent paper (Wang et al., 2008), Zhu and colleagues overexpressed amyloid precursor protein (APP) in human neuroblastoma cells and found changes in mitochondrial morphology and distribution that contributed to neuronal dysfunction.

Interestingly, the Chinese researchers found that while the Aβ-overexpressing flies had lower numbers of axonal mitochondria, those fewer mitochondria were larger than normal. The authors took this as a hint that Aβ may have induced mitochondrial fission defects in the flies. However, other scientists caution that this is pure speculation. “It could simply be caused by damage of mitochondria by Aβ and subsequent removal of abnormal mitochondria,” Lu wrote. Zhu agrees, noting that “without detailed characterization of the changes in mitochondrial fission/fusion machinery, it is probably premature at this stage to suggest impaired mitochondrial fission in this model because swelled mitochondria have been found in cells with mitochondrial fusion defects and during cell death (Chen et al., 2007). Zhu pointed out that Pink1 and parkin mutations have had conflicting effects on mitochondrial fission—impairing it in fly models, yet enhancing it in mammalian cells (see full comment below). In any case, Zhu notes, the current findings confirm a previous study of Aβ42-overexpressing flies (Iijima-Ando et al., 2009) and support the idea that Aβ-induced mitochondrial distribution abnormalities can lead to synaptic deficits in vivo.—Esther Landhuis.

References:
Narendra DP, Jin SM, Tanaka A, Suen DF, Gautier CA, Shen J, Cookson MR, Youle RJ. PINK1 is Selectively Stabilized on Impaired Mitochondria to Activate Parkin. PLoS Biol. 2010 Jan 26;8(1):e1000298. Abstract

Geisler S, Holmstroem KM, Skujat D, Fiesel FC, Rothfuss OC, Kahle PJ, Springer W. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nature Cell Biology. 2010 Jan 24. Abstract

Zhao XL, Wang WA, Tan JX, Huang JK, Zhang X, Zhang BZ, Wang YH, YangCheng HY, Zhu HL, Sun XJ, Huang FD. Expression of beta-amyloid induced age-dependent presynaptic and axonal changes in Drosophila. J Neurosci. 2010 Jan 27;30(4):1512-1522. Abstract

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Comments on News and Primary Papers

  1. The authors described a new system to examine the effect of Aβ on neuronal morphogenesis and physiology in the adult fly brain. They observed depletion of mitochondria in neuronal processes in aged brain. Although the authors raised the possibility of mitochondrial fission defects induced by Aβ as a possible cause, it is pure speculation at this point. It could simply be caused by damage of mitochondria by Aβ and subsequent removal of abnormal mitochondria. An earlier study published in PNAS (Wang et al., 2008) provided more convincing evidence linking Aβ to mitochondrial fission/fusion defects in mammalian hippocampal neurons.

    View all comments by Bingwei Lu
  2. We thank Dr. Zhu for citing our work (Iijima-Ando et al., 2009) in his comment. In our Aβ42 fly brain neurons, mitochondria were reduced in axons and dendrites, and accumulated in the somata without severe mitochondrial damage or neurodegeneration. At this stage, organization of microtubules and distribution of synaptic vesicle markers were not significantly altered, suggesting that mitochondrial mislocalization occurs without global axonal transport defects.

    By knocking down milton, an adaptor protein that links mitochondria and kinesin, we showed that reduction in mitochondria transport exacerbated Aβ42-induced behavioral defects. Furthermore, milton knockdown by itself caused neuronal dysfunction at a later stage. Our results indicate that Aβ42-induced mitochondrial mislocalization contributes to Aβ42-induced neuronal dysfunction in vivo.

    References:

    . Mitochondrial mislocalization underlies Abeta42-induced neuronal dysfunction in a Drosophila model of Alzheimer's disease. PLoS One. 2009;4(12):e8310. PubMed.

  3. The authors did a marvelous job in providing biochemical and cell biological evidence linking the Pink1/parkin pathway to mitochondrial autophagy. This adds significant new insights into the mechanisms by which the Pink1/parkin pathway regulates mitochondrial morphology and function. What remains to be determined, though, is the in vivo relevance of the findings to dopaminergic neuron maintenance and survival. This is particularly important given recent studies in Drosophila that argue against a role of autophagy in Pink1/parkin pathogenesis (Tain et al., 2009).

    View all comments by Bingwei Lu
  4. Synaptic loss and mitochondrial dysfunction are both early features of Alzheimer disease, and connecting the two, as demonstrated in several recent studies including this present work, is attractive. Our recent work revealed that mitochondria accumulate in the soma and are reduced in neuronal processes in AD pyramidal neurons (Wang et al., 2009). To explore the functional consequence of mitochondrial redistribution, we were able to demonstrate that overexpression of APP (Wang et al., 2008) or exposure to soluble Aβ oligomers (Wang et al., 2009) led to reduced neuritic mitochondrial density which correlated with reduced spine number and PSD95-positive puncta. More importantly, repopulation of neurites with mitochondria by overexpressing DLP1 in these cell models alleviates synaptic deficits, thus suggesting that abnormal mitochondrial localization is probably the most important contributing factor of synaptic dysfunction in the pathogenesis of AD. Such a notion is strongly supported by evidence presented in this study. Through the use of fly overexpressing Aβ, which demonstrates intracellular accumulation of Aβ in the soma and axon of a small group of neurons, Fu-De Huang’s group identified multiple mitochondrial abnormalities (i.e., depletion of presynaptic and axonal mitochondria, decreased axonal transport of mitochondria, and changes in mitochondrial size and number), along with presynaptic deficits and deficits in motor behavior. Most importantly, the depletion of presynaptic and axonal mitochondria was the earliest detectable phenotype, which placed abnormal mitochondrial distribution likely upstream to Aβ-induced presynaptic deficits and deficits in motor function. Interestingly, Aβ-induced mitochondrial mislocalization is also confirmed in another Aβ-overexpressing fly model (Iijima-Ando et al., 2009). These studies thus provide compelling evidence to support the notion that Aβ-induced abnormal mitochondrial distribution causes synaptic deficits in vivo.

    Early work from Mark Smith and George Perry’s group (Hirai et al., 2001) demonstrated increased ultrastructural damage to mitochondria in susceptible pyramidal neurons in AD brain. They also observed increased size and decreased number of mitochondria in these neurons. These morphometric changes in AD brain were faithfully replicated in Fu-De Huang’s fly model of AD. Given that mitochondrial number and morphology are strictly regulated by the delicate balance of mitochondrial fission/fusion, these findings likely suggest alteration in mitochondrial dynamics. However, without detailed characterization of the changes in mitochondrial fission/fusion machinery, it is probably premature at this stage to suggest impairment in mitochondrial fission in this model because swelling mitochondria were found in cells deficient of mitochondrial fusion (Chen et al., 2007) and during cell death. In fact, neuronal cells expressing APP or exposed to soluble Aβ oligomers demonstrated enhanced mitochondrial fission through differential effects on the expression of mitochondrial fission/fusion proteins and increased phosphorylation, S-nitrosylation, and mitochondrial recruitment of DLP1 (Wang et al., 2009; Cho et al., 2009). Nevertheless, it remains to be determined whether Aβ/APP overexpression leads to mitochondrial fission in vivo. It is of interest to note that Pink1/parkin mutations cause impaired fission in fly models (Poole et al., 2008; Yang et al., 2008; Deng et al., 2008) while enhancing mitochondrial fission in mammalian cells (Dagda et al., 2009; Lutz et al., 2009; Sandebring et al., 2009); therefore, it may still be possible that overexpression of Aβ/APP induces different effects in fly and mouse models.

    It was reported that Aβ fibrils caused acute impairment in axonal transport of mitochondria. More recently, we also reported that soluble Aβ oligomers caused reduced axonal transport of mitochondria in primary hippocampal neurons, similarly affecting both anterograde and retrograde transport. Consistent with these studies, reduced axonal transport of mitochondria is also reported in this current study. Since it occurs later than the presynaptic depletion of mitochondria, the authors suggest that axonal transport deficits may not cause mitochondrial depletion. However, another recent study in a similar Aβ-overexpressing fly model suggests that mitochondrial transport likely contributes to the mislocalization of mitochondria (Iijima-Ando et al., 2009). Therefore, more studies, especially those in mammalian systems, are still needed.

    View all comments by Xiongwei Zhu
  5. For mitochondrial effects in AD please see our Pharmacogenomics Journal article published in 2009. localizing a variable polyT mutation in the translocase of the outer mitochondrial membrane as a diagnostic predictor of risk for AD.

  6. We thank all the commentators on our paper, "Expression of beta amyloid induced age-dependent presynaptic and axonal changes in Drosophila."

    Our examination, through genetic manipulation, of the role of critical mitochondria fission and fusion genes in the mitochondrial abnormalities induced by Aβ expression will be finished soon.

References

News Citations

  1. Pink Fission—Serving Up a Rationale for Parkinson Disease?
  2. Mitochondrial Break-up: Alzheimer’s Alters Fusion, Fission

Paper Citations

  1. . Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature. 2006 Jun 29;441(7097):1162-6. PubMed.
  2. . 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.
  3. . Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature. 2006 Jun 29;441(7097):1157-61. PubMed.
  4. . Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol. 2008 Dec 1;183(5):795-803. PubMed.
  5. . Impaired balance of mitochondrial fission and fusion in Alzheimer's disease. J Neurosci. 2009 Jul 15;29(28):9090-103. PubMed.
  6. . Amyloid-beta overproduction causes abnormal mitochondrial dynamics via differential modulation of mitochondrial fission/fusion proteins. Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19318-23. PubMed.
  7. . Mitochondrial fusion protects against neurodegeneration in the cerebellum. Cell. 2007 Aug 10;130(3):548-62. PubMed.
  8. . Mitochondrial mislocalization underlies Abeta42-induced neuronal dysfunction in a Drosophila model of Alzheimer's disease. PLoS One. 2009;4(12):e8310. PubMed.
  9. . PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 2010 Jan;8(1):e1000298. PubMed.
  10. . PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol. 2010 Feb;12(2):119-31. PubMed.
  11. . Expression of beta-amyloid induced age-dependent presynaptic and axonal changes in Drosophila. J Neurosci. 2010 Jan 27;30(4):1512-22. PubMed.

Further Reading

Papers

  1. . Impaired balance of mitochondrial fission and fusion in Alzheimer's disease. J Neurosci. 2009 Jul 15;29(28):9090-103. PubMed.
  2. . Amyloid-beta overproduction causes abnormal mitochondrial dynamics via differential modulation of mitochondrial fission/fusion proteins. Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19318-23. PubMed.
  3. . S-nitrosylation of Drp1 mediates beta-amyloid-related mitochondrial fission and neuronal injury. Science. 2009 Apr 3;324(5923):102-5. 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. . Cytoplasmic Pink1 activity protects neurons from dopaminergic neurotoxin MPTP. Proc Natl Acad Sci U S A. 2008 Feb 5;105(5):1716-21. PubMed.
  6. . 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.
  7. . PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 2010 Jan;8(1):e1000298. PubMed.
  8. . PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol. 2010 Feb;12(2):119-31. PubMed.
  9. . Expression of beta-amyloid induced age-dependent presynaptic and axonal changes in Drosophila. J Neurosci. 2010 Jan 27;30(4):1512-22. PubMed.

Primary Papers

  1. . PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 2010 Jan;8(1):e1000298. PubMed.
  2. . PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol. 2010 Feb;12(2):119-31. PubMed.
  3. . Expression of beta-amyloid induced age-dependent presynaptic and axonal changes in Drosophila. J Neurosci. 2010 Jan 27;30(4):1512-22. PubMed.