This article was downloaded by: [Michigan State University] On: 28 February 2015, At: 04:04 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Autophagy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kaup20
PARK2/Parkin becomes critical when DNM1L/Drp1 is absent a
a
a
Madhuparna Roy , Yusuke Kageyama , Miho Iijima & Hiromi Sesaki
a
a
Department of Cell Biology; Johns Hopkins University School of Medicine; Baltimore, MD USA Accepted author version posted online: 25 Feb 2015.
Click for updates To cite this article: Madhuparna Roy, Yusuke Kageyama, Miho Iijima & Hiromi Sesaki (2015): PARK2/Parkin becomes critical when DNM1L/Drp1 is absent, Autophagy To link to this article: http://dx.doi.org/10.1080/15548627.2015.1017193
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
PARK2/Parkinbecomescriticalwhen DNM1L/Drp1 is absent
Madhuparna Roy, Yusuke Kageyama, Miho Iijima, and Hiromi Sesaki* Department of Cell Biology; Johns Hopkins University School of Medicine; Baltimore, MD
ip t
USA
*Correspondence to: Hiromi Sesaki; Email: [email protected]
cr
us
sustainingcellular health. Defects in these processes have been linked to cardiovascular diseases and neurodegeneration. In a recent publication, we reported that the mitochondrial division dynaminprotein DNM1L/Drp1 and the E3 ubiquitin ligase PARK2/Parkin work in a synergistic
an
manner to maintain mitochondrial function and structural integrity in the mouse heart and brain.
ce
pt
ed
M
Keywords: Drp1, mitochondrial dynamics, mitochondrial homeostasis, mitophagy, Parkin.
Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
Maintaining mitochondrial dynamics and proper execution of mitophagy is crucial for
1
Mitochondria are highly dynamic structures that constantly divide and fuse to meet cellular energy demands. High-energy consuming cells found in the heart and brainare especially rich in mitochondria.Dysfunctional mitochondrial division and fusion processes in thesecells may lead to heart abnormalities and neurodegenerative disorders.Recently, weinvestigated the role of mitochondrial dynamics in2 cell types:cardiomyocytes intheheart that have low
ip t
mitochondrial division rates and Purkinje neurons (PNs)inthe cerebellum, which have high mitochondrial division rates.
To understand the role of DNM1L in the heart, mice lacking DNM1L specificallyin the
cr
us
with Dnm1lflox mice, leading to the loss of DNM1L during the early postnatal days.
DNM1Ldeficiency results in decreased heart contraction and beat rate, ultimately causing lethality between P9 and P11. Thus, DNM1L is critically important for sustaining heart function
an
and animal survival. In these animals heart failure is likely caused by decreased mitochondrial respiration in Dnm1l knockout (KO)cardiomyocytes. Histological activity assays, oxygen
M
consumption measurements, staining and biochemical activity analysis revealed a decrease in the activity of the electron transport chain complexes in such cells. To determine the effect of a lack of mitochondrial division on
ed
cardiomyocytemorphology, we characterized the ultrastructure of control and Dnm1lKOcardiomyocytes using conventional transmission electron microscopy, tomographic
pt
electron microscopy,and immunofluorescence microscopy.We observed that control cardiomyocyteshave short tubular mitochondria whereasDnm1lKO cardiomyocyteshave
ce
interconnected and enlarged spherical mitochondria. Interestingly, oxidized proteins and lipids are enriched in the enlarged spherical mitochondria of Dnm1lKO cardiomyocytesas revealed by immunofluorescence microscopy using anti-HNE antibodies. In contrast, the elongated tubular
Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
heart were generatedusing the Crerecombinase system. MyH6-Cre transgenic mice were bred
mitochondria do not appearto accumulate oxidative damage. These observations together with our previous studies suggest that large spherical mitochondria likely result from acombination of defective mitochondrial division and an accumulation of oxidative damage. Although mitochondrial morphology is altered in Dnm1l KO cardiomyocytes, no activation of cell death pathways isdetected. To understand why mitochondria accumulate oxidative damage inDnm1lKO cardiomyocytes, we examined mitophagy. We found striking accumulation of a 2
mitophagyreceptor protein, SQSTM1/p62,on the surface of enlarged spherical mitochondriaalong with ubiquitinated proteins.Interestingly, no difference is observed in LC3-II levels. It is likely that mitophagyis incompletely executed in Dnm1lKO cardiomyocytesbecause the ubiquitin-tagged mitochondria do not colocalize with lysosomes. We suggest that mitochondrial division deficiency leads to inefficient mitophagy, causing accumulation of
ip t
mitochondria with oxidative damage and respiration defects. The loss of function likely promotes further attempts of mitophagy and recruitment of ubiquitin E3 ligases and SQSTM1 to damaged mitochondria.
cr
us
ubiquitinates mitochondrial outer membrane proteins. Defects in PARK2 can cause sporadic and familial Parkinson disease. Therefore, understanding the function of PARK2has recently become a high priority in the field of mitochondrial quality control. To test the involvement of
an
PARK2 in mitochondrial protein ubiquitination in Dnm1l KO cardiomyocytes, we crossed Park2KO mice with Myh6-Dnm1lKO mice. Surprisingly, we found that
M
mitochondrialubiquitinationand SQSTM1 accumulation are not decreased in Dnm1l Park2 double-KO cardiomyocytes. These results led to at least 2 hypotheses. First, PARK2 is not involved in mitophagy that is promoted by DNM1L-mediated mitochondrial division.Second,
ed
PARK2 does function in DNM1L-dependent mitophagy, but in the absence of PARK2 other E3 ligases functionally compensate for its loss. Supporting the first hypothesis, when we analyzed
pt
the effect of dual loss of PARK2 and DNM1L, both defects in mitochondrial respiration and heart function increase in the double-knockout mice.Park2KO mice do not have cardiac defects
ce
or neurodegenerative phenotypes; however, upon the loss of mitochondrial division, PARK2 becomes critical for the maintenance of mitochondrial integrity and heart function. To determine how general our findings are, we also generated double-knockout PARK2
Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
PARK2 is an E3 ubiquitin ligase that is recruited to dysfunctional mitochondria and
and DNM1Lcerebellar Purkinje neurons (PNs)in vivo. Understanding the functional relationship between DNM1L and PARK2 in neurons is very important because defects in PARK2 cause neurodegenerativeParkinson disease. We have previously shown that the loss of DNM1L causes mitochondrial respiratory defects and neurodegeneration in PNs. Our new studyshows thatthese
phenotypesare exacerbated by the additional loss of PARK2, that is, in Dnm1l Park2KO PNs, although the single Park2KOPNs are indistinguishable from control PNs. These results further support the notion that DNM1Land PARK2are necessary for maintaining mitochondrial 3
functionand neuronal survival in a synergistic fashion.PARK2 becomes critical when mitochondrial division is lost, suggesting that the onset and progression of Parkinson disease may correlate with age-dependent decreases in mitochondrial division. Accumulation of mitochondrial ubiquitination in the absence of DNM1L appear to be cell-type specific. We observe no accumulation of ubiquitinated proteins on mitochondria in
ip t
Dnm1lKO embryonic fibroblasts. This could be explained by several mechanisms including decreased ubiquitination activity, rapid degradation of ubiquitinated proteins by proteasomes, removal of ubiquitin by deubiquitinating enzymes, or DNM1L-independent mitophagy.
cr
us
embryonic fibroblastsby using a biosensor called mito-Keima, which measures the delivery of mitochondria to lysosomesusing live cell imaging. Consistent with this, Dnm1l KO mouse embryonic fibroblastsshow normal mitochondrial respiration. However, similar to
an
cardiomyocytes and PNs, when both DNM1L and PARK2 are absent, mitophagy dramatically decreases and mitochondrial respiration is strikingly lowered, resulting in defects in cell
M
proliferation.Furthermore, mitochondrial DNA is aggregated in the portion of mitochondria with enlarged morphology. Therefore, synergistic roles of DNM1L and PARK2 are universal among different cell types.
ed
It has been proposed that PARK2ubiquitinates mitochondrial proteins,whereasDNM1L initiates mitochondrial division, thereby stimulating engulfment of defective mitochondria within
pt
phagophores. Our new study suggests a model in which there are at least 2 types of mitophagy; a DNM1L-dependent pathway and a PARK2-dependent pathway (Fig. 1). These pathways
ce
synergistically function in the homeostasis of mitochondria and in cell proliferation, function, and survival.Our data hint at the existence of novel E3 ligasesinvolved in the DNM1L-dependent mitophagy pathway.We also suggest that the PARK2-dependent pathway involves DNM1L-
Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
Supportive of the last model, we found that mitophagyis mildly decreased in Dnm1l KO mouse
independent mitochondrial division. It would be of great interest to determinehow these 2 pathwaysinteract. Another important mechanistic question is whichmitochondrial proteins are ubiquitinated in DNM1L-dependent mitophagy and what is the function of their ubiquitination. In our future studies, we hope to identify these key components to significantly advance our understanding of the DNM1L-dependent mitophagy pathway.
Acknowledgements 4
This work was supported by NIH grants GM084015 (MI), GM089853 (HS), and NS084154
cr us an M ed pt ce Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
ip t
(HS) and by the Mirowski Discovery Fund (HS).
5
ip t cr us an
M
DNM1L-dependent mitophagy. Twomitophagypathwaysynergistically maintain mitochondrial homeostasis and cellular function and viability; each can operate in part independently, but they
ce
pt
ed
also overlap.
Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
Figure 1.Model for 2 interacting mitophagy pathways: PARK2-dependent mitophagy and
6
Autophagy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kaup20
PARK2/Parkin becomes critical when DNM1L/Drp1 is absent a
a
a
Madhuparna Roy , Yusuke Kageyama , Miho Iijima & Hiromi Sesaki
a
a
Department of Cell Biology; Johns Hopkins University School of Medicine; Baltimore, MD USA Accepted author version posted online: 25 Feb 2015.
Click for updates To cite this article: Madhuparna Roy, Yusuke Kageyama, Miho Iijima & Hiromi Sesaki (2015): PARK2/Parkin becomes critical when DNM1L/Drp1 is absent, Autophagy To link to this article: http://dx.doi.org/10.1080/15548627.2015.1017193
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
PARK2/Parkinbecomescriticalwhen DNM1L/Drp1 is absent
Madhuparna Roy, Yusuke Kageyama, Miho Iijima, and Hiromi Sesaki* Department of Cell Biology; Johns Hopkins University School of Medicine; Baltimore, MD
ip t
USA
*Correspondence to: Hiromi Sesaki; Email: [email protected]
cr
us
sustainingcellular health. Defects in these processes have been linked to cardiovascular diseases and neurodegeneration. In a recent publication, we reported that the mitochondrial division dynaminprotein DNM1L/Drp1 and the E3 ubiquitin ligase PARK2/Parkin work in a synergistic
an
manner to maintain mitochondrial function and structural integrity in the mouse heart and brain.
ce
pt
ed
M
Keywords: Drp1, mitochondrial dynamics, mitochondrial homeostasis, mitophagy, Parkin.
Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
Maintaining mitochondrial dynamics and proper execution of mitophagy is crucial for
1
Mitochondria are highly dynamic structures that constantly divide and fuse to meet cellular energy demands. High-energy consuming cells found in the heart and brainare especially rich in mitochondria.Dysfunctional mitochondrial division and fusion processes in thesecells may lead to heart abnormalities and neurodegenerative disorders.Recently, weinvestigated the role of mitochondrial dynamics in2 cell types:cardiomyocytes intheheart that have low
ip t
mitochondrial division rates and Purkinje neurons (PNs)inthe cerebellum, which have high mitochondrial division rates.
To understand the role of DNM1L in the heart, mice lacking DNM1L specificallyin the
cr
us
with Dnm1lflox mice, leading to the loss of DNM1L during the early postnatal days.
DNM1Ldeficiency results in decreased heart contraction and beat rate, ultimately causing lethality between P9 and P11. Thus, DNM1L is critically important for sustaining heart function
an
and animal survival. In these animals heart failure is likely caused by decreased mitochondrial respiration in Dnm1l knockout (KO)cardiomyocytes. Histological activity assays, oxygen
M
consumption measurements, staining and biochemical activity analysis revealed a decrease in the activity of the electron transport chain complexes in such cells. To determine the effect of a lack of mitochondrial division on
ed
cardiomyocytemorphology, we characterized the ultrastructure of control and Dnm1lKOcardiomyocytes using conventional transmission electron microscopy, tomographic
pt
electron microscopy,and immunofluorescence microscopy.We observed that control cardiomyocyteshave short tubular mitochondria whereasDnm1lKO cardiomyocyteshave
ce
interconnected and enlarged spherical mitochondria. Interestingly, oxidized proteins and lipids are enriched in the enlarged spherical mitochondria of Dnm1lKO cardiomyocytesas revealed by immunofluorescence microscopy using anti-HNE antibodies. In contrast, the elongated tubular
Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
heart were generatedusing the Crerecombinase system. MyH6-Cre transgenic mice were bred
mitochondria do not appearto accumulate oxidative damage. These observations together with our previous studies suggest that large spherical mitochondria likely result from acombination of defective mitochondrial division and an accumulation of oxidative damage. Although mitochondrial morphology is altered in Dnm1l KO cardiomyocytes, no activation of cell death pathways isdetected. To understand why mitochondria accumulate oxidative damage inDnm1lKO cardiomyocytes, we examined mitophagy. We found striking accumulation of a 2
mitophagyreceptor protein, SQSTM1/p62,on the surface of enlarged spherical mitochondriaalong with ubiquitinated proteins.Interestingly, no difference is observed in LC3-II levels. It is likely that mitophagyis incompletely executed in Dnm1lKO cardiomyocytesbecause the ubiquitin-tagged mitochondria do not colocalize with lysosomes. We suggest that mitochondrial division deficiency leads to inefficient mitophagy, causing accumulation of
ip t
mitochondria with oxidative damage and respiration defects. The loss of function likely promotes further attempts of mitophagy and recruitment of ubiquitin E3 ligases and SQSTM1 to damaged mitochondria.
cr
us
ubiquitinates mitochondrial outer membrane proteins. Defects in PARK2 can cause sporadic and familial Parkinson disease. Therefore, understanding the function of PARK2has recently become a high priority in the field of mitochondrial quality control. To test the involvement of
an
PARK2 in mitochondrial protein ubiquitination in Dnm1l KO cardiomyocytes, we crossed Park2KO mice with Myh6-Dnm1lKO mice. Surprisingly, we found that
M
mitochondrialubiquitinationand SQSTM1 accumulation are not decreased in Dnm1l Park2 double-KO cardiomyocytes. These results led to at least 2 hypotheses. First, PARK2 is not involved in mitophagy that is promoted by DNM1L-mediated mitochondrial division.Second,
ed
PARK2 does function in DNM1L-dependent mitophagy, but in the absence of PARK2 other E3 ligases functionally compensate for its loss. Supporting the first hypothesis, when we analyzed
pt
the effect of dual loss of PARK2 and DNM1L, both defects in mitochondrial respiration and heart function increase in the double-knockout mice.Park2KO mice do not have cardiac defects
ce
or neurodegenerative phenotypes; however, upon the loss of mitochondrial division, PARK2 becomes critical for the maintenance of mitochondrial integrity and heart function. To determine how general our findings are, we also generated double-knockout PARK2
Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
PARK2 is an E3 ubiquitin ligase that is recruited to dysfunctional mitochondria and
and DNM1Lcerebellar Purkinje neurons (PNs)in vivo. Understanding the functional relationship between DNM1L and PARK2 in neurons is very important because defects in PARK2 cause neurodegenerativeParkinson disease. We have previously shown that the loss of DNM1L causes mitochondrial respiratory defects and neurodegeneration in PNs. Our new studyshows thatthese
phenotypesare exacerbated by the additional loss of PARK2, that is, in Dnm1l Park2KO PNs, although the single Park2KOPNs are indistinguishable from control PNs. These results further support the notion that DNM1Land PARK2are necessary for maintaining mitochondrial 3
functionand neuronal survival in a synergistic fashion.PARK2 becomes critical when mitochondrial division is lost, suggesting that the onset and progression of Parkinson disease may correlate with age-dependent decreases in mitochondrial division. Accumulation of mitochondrial ubiquitination in the absence of DNM1L appear to be cell-type specific. We observe no accumulation of ubiquitinated proteins on mitochondria in
ip t
Dnm1lKO embryonic fibroblasts. This could be explained by several mechanisms including decreased ubiquitination activity, rapid degradation of ubiquitinated proteins by proteasomes, removal of ubiquitin by deubiquitinating enzymes, or DNM1L-independent mitophagy.
cr
us
embryonic fibroblastsby using a biosensor called mito-Keima, which measures the delivery of mitochondria to lysosomesusing live cell imaging. Consistent with this, Dnm1l KO mouse embryonic fibroblastsshow normal mitochondrial respiration. However, similar to
an
cardiomyocytes and PNs, when both DNM1L and PARK2 are absent, mitophagy dramatically decreases and mitochondrial respiration is strikingly lowered, resulting in defects in cell
M
proliferation.Furthermore, mitochondrial DNA is aggregated in the portion of mitochondria with enlarged morphology. Therefore, synergistic roles of DNM1L and PARK2 are universal among different cell types.
ed
It has been proposed that PARK2ubiquitinates mitochondrial proteins,whereasDNM1L initiates mitochondrial division, thereby stimulating engulfment of defective mitochondria within
pt
phagophores. Our new study suggests a model in which there are at least 2 types of mitophagy; a DNM1L-dependent pathway and a PARK2-dependent pathway (Fig. 1). These pathways
ce
synergistically function in the homeostasis of mitochondria and in cell proliferation, function, and survival.Our data hint at the existence of novel E3 ligasesinvolved in the DNM1L-dependent mitophagy pathway.We also suggest that the PARK2-dependent pathway involves DNM1L-
Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
Supportive of the last model, we found that mitophagyis mildly decreased in Dnm1l KO mouse
independent mitochondrial division. It would be of great interest to determinehow these 2 pathwaysinteract. Another important mechanistic question is whichmitochondrial proteins are ubiquitinated in DNM1L-dependent mitophagy and what is the function of their ubiquitination. In our future studies, we hope to identify these key components to significantly advance our understanding of the DNM1L-dependent mitophagy pathway.
Acknowledgements 4
This work was supported by NIH grants GM084015 (MI), GM089853 (HS), and NS084154
cr us an M ed pt ce Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
ip t
(HS) and by the Mirowski Discovery Fund (HS).
5
ip t cr us an
M
DNM1L-dependent mitophagy. Twomitophagypathwaysynergistically maintain mitochondrial homeostasis and cellular function and viability; each can operate in part independently, but they
ce
pt
ed
also overlap.
Ac
Downloaded by [Michigan State University] at 04:04 28 February 2015
Figure 1.Model for 2 interacting mitophagy pathways: PARK2-dependent mitophagy and
6
