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Dynamin-Related Protein 1 Deficiency Leads to Receptor-Interacting Protein Kinase 3–Mediated Necroptotic Neurodegeneration

2016; Elsevier BV; Volume: 186; Issue: 11 Linguagem: Inglês

10.1016/j.ajpath.2016.06.025

1525-2191

Tatsuya Yamada, Yoshihiro Adachi, Masahiro Fukaya, Miho Iijima, Hiromi Sesaki,

Cellular transport and secretion

Mitochondria are dynamic organelles that divide and fuse to modulate their number and shape. We have previously reported that the loss of dynamin-related protein 1 (Drp1), which mediates mitochondrial division, leads to the degeneration of cerebellar Purkinje cells in mice. Because Drp1 has been shown to be important for apoptosis and necroptosis, it is puzzling how Purkinje neurons die in the absence of Drp1. In this study, we tested whether neurodegeneration involves necrotic cell death by generating Purkinje cell–specific Drp1-knockout (KO) mice that lack the receptor-interacting protein kinase 3 (Rip3), which regulates necroptosis. We found that the loss of Rip3 significantly delays the degeneration of Drp1-KO Purkinje neurons. In addition, before neurodegeneration, mitochondrial tubules elongate because of unopposed fusion and subsequently become large spheres as a result of oxidative damage. Surprisingly, Rip3 loss also helps Drp1-KO Purkinje cells maintain the elongated morphology of the mitochondrial tubules. These data suggest that Rip3 plays a role in neurodegeneration and mitochondrial morphology in the absence of mitochondrial division. Mitochondria are dynamic organelles that divide and fuse to modulate their number and shape. We have previously reported that the loss of dynamin-related protein 1 (Drp1), which mediates mitochondrial division, leads to the degeneration of cerebellar Purkinje cells in mice. Because Drp1 has been shown to be important for apoptosis and necroptosis, it is puzzling how Purkinje neurons die in the absence of Drp1. In this study, we tested whether neurodegeneration involves necrotic cell death by generating Purkinje cell–specific Drp1-knockout (KO) mice that lack the receptor-interacting protein kinase 3 (Rip3), which regulates necroptosis. We found that the loss of Rip3 significantly delays the degeneration of Drp1-KO Purkinje neurons. In addition, before neurodegeneration, mitochondrial tubules elongate because of unopposed fusion and subsequently become large spheres as a result of oxidative damage. Surprisingly, Rip3 loss also helps Drp1-KO Purkinje cells maintain the elongated morphology of the mitochondrial tubules. These data suggest that Rip3 plays a role in neurodegeneration and mitochondrial morphology in the absence of mitochondrial division. Dynamin-related protein 1 (Drp1) plays a critical role in mitochondrial division by limiting mitochondria as a mechanochemical GTPase.1Roy M. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division and fusion in metabolism.Curr Opin Cell Biol. 2015; 33C: 111-118Crossref Scopus (139) Google Scholar, 2Bui H.T. Shaw J.M. Dynamin assembly strategies and adaptor proteins in mitochondrial fission.Curr Biol. 2013; 23: R891-R899Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 3Tamura Y. Itoh K. Sesaki H. 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Sesaki H. The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice.J Cell Biol. 2009; 186: 805-816Crossref PubMed Scopus (462) Google Scholar, 13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar, 14Kageyama Y. Hoshijima M. Seo K. Bedja D. Sysa-Shah P. Andrabi S.A. Chen W. Hoke A. Dawson V.L. Dawson T.M. Gabrielson K. Kass D.A. Iijima M. Sesaki H. Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain.EMBO J. 2014; 33: 2798-2813Crossref PubMed Scopus (299) Google Scholar, 15Shields L.Y. Kim H. Zhu L. Haddad D. Berthet A. Pathak D. Lam M. Ponnusamy R. Diaz-Ramirez L.G. Gill T.M. Sesaki H. Mucke L. Nakamura K. Dynamin-related protein 1 is required for normal mitochondrial bioenergetic and synaptic function in CA1 hippocampal neurons.Cell Death Dis. 2015; 6: e1725Crossref PubMed Scopus (65) Google Scholar, 16Berthet A. Margolis E.B. Zhang J. Hsieh I. Hnasko T.S. Ahmad J. Edwards R.H. Sesaki H. Huang E.J. Nakamura K. Loss of mitochondrial fission depletes axonal mitochondria in midbrain dopamine neurons.J Neurosci. 2014; 34: 14304-14317Crossref PubMed Scopus (133) Google Scholar Complete loss of Drp1 causes lethality around embryonic day 11.5,12Wakabayashi J. Zhang Z. Wakabayashi N. Tamura Y. Fukaya M. Kensler T.W. Iijima M. Sesaki H. The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice.J Cell Biol. 2009; 186: 805-816Crossref PubMed Scopus (462) Google Scholar, 17Ishihara N. Nomura M. Jofuku A. Kato H. Suzuki S.O. Masuda K. Otera H. Nakanishi Y. Nonaka I. Goto Y. Taguchi N. Morinaga H. Maeda M. Takayanagi R. Yokota S. Mihara K. Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice.Nat Cell Biol. 2009; 11: 958-966Crossref PubMed Scopus (756) Google Scholar demonstrating the importance of Drp1 in embryonic development. Similar to human patients carrying mutations in Drp1, the loss of Drp1 in the developing animal cerebellum leads to severe defects in cerebellar development with decreased cell proliferation and postnatal death.12Wakabayashi J. Zhang Z. Wakabayashi N. Tamura Y. Fukaya M. Kensler T.W. Iijima M. Sesaki H. The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice.J Cell Biol. 2009; 186: 805-816Crossref PubMed Scopus (462) Google Scholar Importantly, the physiological impacts of Drp1 loss in these mice vary, depending on neuron type. Purkinje neurons, which express high levels of Drp1, display dramatic increases in the size of their mitochondria because of unopposed mitochondrial fusion in the absence of division in the Drp1-KO cerebellum.12Wakabayashi J. Zhang Z. Wakabayashi N. Tamura Y. Fukaya M. Kensler T.W. Iijima M. Sesaki H. The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice.J Cell Biol. 2009; 186: 805-816Crossref PubMed Scopus (462) Google Scholar In contrast, granule neurons, which only modestly express Drp1, exhibit normal mitochondrial morphology in the same Drp1-KO cerebellum. Drp1 is required for the maintenance of post-mitotic Purkinje neurons. To circumvent the developmental effects and study the role of Drp1 in post-mitotic neurons, we deleted Drp1 in post-mitotic Purkinje neurons after the completion of development using Cre recombinase, which is expressed from the Purkinje cell–specific L7 promoter.13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar, 14Kageyama Y. Hoshijima M. Seo K. Bedja D. Sysa-Shah P. Andrabi S.A. Chen W. Hoke A. Dawson V.L. Dawson T.M. Gabrielson K. Kass D.A. Iijima M. Sesaki H. Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain.EMBO J. 2014; 33: 2798-2813Crossref PubMed Scopus (299) Google Scholar Drp1-KO Purkinje neurons produce excessively elongated mitochondrial tubules because of a lack of division; these long tubules then unexpectedly collapse into large spherical structures because of the accumulation of oxidative damage from decreased mitophagy.13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar These mitochondrial defects result in respiratory deficiencies and the progressive degeneration of Drp1-KO Purkinje neurons.13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar It has been shown that decreasing mitochondrial size and remodeling of the mitochondrial outer membrane facilitates BAX oligomerization and cytochrome c release from mitochondria during apoptosis.18Montessuit S. Somasekharan S.P. Terrones O. Lucken-Ardjomande S. Herzig S. Schwarzenbacher R. Manstein D.J. Bossy-Wetzel E. Basanez G. Meda P. Martinou J.C. Membrane remodeling induced by the dynamin-related protein Drp1 stimulates Bax oligomerization.Cell. 2010; 142: 889-901Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 19Renault T.T. Floros K.V. Elkholi R. Corrigan K.A. Kushnareva Y. Wieder S.Y. Lindtner C. Serasinghe M.N. Asciolla J.J. Buettner C. Newmeyer D.D. Chipuk J.E. Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis.Mol Cell. 2015; 57: 69-82Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar During degeneration, there were no signs of apoptotic cell death in Drp1-KO Purkinje neurons, consistent with the role of Drp1 in apoptosis as a proapoptotic factor.13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar Interestingly, in addition to apoptosis, recent studies have suggested that Drp1-mediated mitochondrial division is also important for necroptosis that is mediated by the protein complex containing receptor-interacting protein kinase 1 and 3 (Rip1 and Rip3, respectively).20Dashzeveg N. Yoshida K. Cell death decision by p53 via control of the mitochondrial membrane.Cancer Lett. 2015; 367: 108-112Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 21Humphries F. Yang S. Wang B. Moynagh P.N. RIP kinases: key decision makers in cell death and innate immunity.Cell Death Differ. 2015; 22: 225-236Crossref PubMed Scopus (167) Google Scholar, 22Zhou W. Yuan J. Necroptosis in health and diseases.Semin Cell Dev Biol. 2014; 35: 14-23Crossref PubMed Scopus (289) Google Scholar, 23Moriwaki K. Chan F.K. RIP3: a molecular switch for necrosis and inflammation.Genes Dev. 2013; 27: 1640-1649Crossref PubMed Scopus (249) Google Scholar, 24Zhou Z. Han V. Han J. New components of the necroptotic pathway.Protein Cell. 2012; 3: 811-817Crossref PubMed Scopus (54) Google Scholar Mitochondrial division is stimulated via the dephosphorylation of Drp1 by a protein phosphatase, PGAM5, which functions as a downstream effector of the Rip1-Rip3 complex.21Humphries F. Yang S. Wang B. Moynagh P.N. RIP kinases: key decision makers in cell death and innate immunity.Cell Death Differ. 2015; 22: 225-236Crossref PubMed Scopus (167) Google Scholar, 25Wang Z. Jiang H. Chen S. Du F. Wang X. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways.Cell. 2012; 148: 228-243Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar The critical roles of Drp1 in both apoptosis and necroptosis prompted us to ask how neurons die when Drp1 is absent. In this study, we genetically blocked necroptosis by taking advantage of Rip3 whole-body KO mice26Newton K. Sun X. Dixit V.M. Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4.Mol Cell Biol. 2004; 24: 1464-1469Crossref PubMed Scopus (416) Google Scholar to reveal the mechanism explaining how neurodegeneration results from a lack of Drp1-mediated mitochondrial division. All of the work with animals was conducted according to guidelines established by the Johns Hopkins University Committee on Animal Care and Use. The Drp1flox/flox and Rip3−/− mice have been described previously.12Wakabayashi J. Zhang Z. Wakabayashi N. Tamura Y. Fukaya M. Kensler T.W. Iijima M. Sesaki H. The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice.J Cell Biol. 2009; 186: 805-816Crossref PubMed Scopus (462) Google Scholar, 26Newton K. Sun X. Dixit V.M. Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4.Mol Cell Biol. 2004; 24: 1464-1469Crossref PubMed Scopus (416) Google Scholar The L7-Cre mice were obtained from the Jackson Laboratory (Bar Harbor, ME).27Barski J.J. Dethleffsen K. Meyer M. Cre recombinase expression in cerebellar Purkinje cells.Genesis. 2000; 28: 93-98Crossref PubMed Scopus (136) Google Scholar By breeding Rip3+/−::L7-Cre+/−::Drp1flox/flox mice and Rip3+/−::Drp1flox/flox mice, we generated littermate control (Drp1flox/flox), Rip3-KO (Rip3−/−::Drp1flox/flox), L7-Drp1-KO (L7-Cre+/−::Drp1flox/flox), and double-KO (Rip3−/−::L7-Cre+/−::Drp1flox/flox) mice. The Drp1flox/flox mice are phenotypically wild type.13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar Immunofluorescence microscopy of cerebellar Purkinje cells was performed as previously described13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar with some modifications.14Kageyama Y. Hoshijima M. Seo K. Bedja D. Sysa-Shah P. Andrabi S.A. Chen W. Hoke A. Dawson V.L. Dawson T.M. Gabrielson K. Kass D.A. Iijima M. Sesaki H. Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain.EMBO J. 2014; 33: 2798-2813Crossref PubMed Scopus (299) Google Scholar The mice were fixed by perfusing ice-cold 4% paraformaldehyde in phosphate-buffered saline. The brain was dissected, fixed in 4% paraformaldehyde in phosphate-buffered saline for 2 hours at 4°C, incubated in phosphate-buffered saline containing 30% sucrose overnight, and frozen in optimal cutting temperature compound (VWR, Rangor, PA). The frozen sections were cut, washed in phosphate-buffered saline, and blocked in 10% donkey or sheep serum. The sections were then incubated with antibodies to carbonic anhydrase 8 (Car8-Rb-Af330; Frontier Institute, Hokkaido, Japan) and pyruvate dehydrogenase (ab110333; Abcam, Cambridge, MA), followed by fluorescently labeled secondary antibodies (Alexa 488 anti-rabbit IgG A21206 and Alexa 568 anti-mouse IgG A10037; Invitrogen, Carlsbad, CA). We examined the samples using a Zeiss LSM780 FCS laser scanning confocal microscope equipped with a 10× (0.4 numerical aperture) objective and a Zeiss LSM510-Meta laser scanning confocal microscope equipped with a 100× (1.3 numerical aperture) objective. We generated control, Rip3-KO (Rip3−/−), L7-Drp1-KO (L7-Drp1flox/flox), and double-KO (Rip3−/−::L7-Drp1flox/flox) mice by breeding. Cerebella of mice at an age of 3 months were fixed, dissected, and sectioned around the median line. Cerebellar sections were subjected to immunofluorescence microscopy using antibodies against the Purkinje cell marker carbonic anhydrase 8, and soma were counted to quantify the number of Purkinje cells. Consistent with our previous studies,14Kageyama Y. Hoshijima M. Seo K. Bedja D. Sysa-Shah P. Andrabi S.A. Chen W. Hoke A. Dawson V.L. Dawson T.M. Gabrielson K. Kass D.A. Iijima M. Sesaki H. Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain.EMBO J. 2014; 33: 2798-2813Crossref PubMed Scopus (299) Google Scholar approximately 65% of the Purkinje cells were lost in the L7-Drp1-KO mice compared with the control mice (Figure 1, A and B). In contrast to the L7-Drp1-KO mice, the Rip3-KO mice contained a normal number of Purkinje neurons (Figure 1, A and B). In double-KO mice, we found a significant increase in the number of Purkinje cells compared with the L7-Drp1-KO mice (Figure 1, A and B). These data suggest that a significant population of Drp1-KO Purkinje cells die via a necroptotic mechanism involving Rip3 at 3 months. To further determine the effect of the loss of Rip3 on Drp1-KO Purkinje cells at a later time, we analyzed double-KO mice at 5 months. We found that the number of Purkinje cells had further decreased at 5 months compared to 3 months in double-KO mice (Supplemental Figure S1). Therefore, Rip3 loss delays the degeneration of Drp1-KO Purkinje cells but does not prevent it. We speculate that other cell death mechanisms, such as apoptosis, may be induced after a prolonged absence of necroptosis to eliminate Purkinje cells that contain dysfunctional mitochondria. This suppression of neurodegeneration via Rip3 loss is interesting for two reasons. First, it has been shown that oxidative stress induces Rip3-mediated necroptosis.22Zhou W. Yuan J. Necroptosis in health and diseases.Semin Cell Dev Biol. 2014; 35: 14-23Crossref PubMed Scopus (289) Google Scholar, 23Moriwaki K. Chan F.K. RIP3: a molecular switch for necrosis and inflammation.Genes Dev. 2013; 27: 1640-1649Crossref PubMed Scopus (249) Google Scholar Second, we have previously shown that feeding L7-Drp1-KO mice an antioxidant, coenzyme Q10, partially rescues the degeneration of Purkinje cells.13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar The rescuing effect of depleting Rip3 on the number of Drp1-KO Purkinje neurons appears to be similar to that of feeding the mice coenzyme Q10.13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar It is possible that the accumulation of reactive oxygen species caused by Drp1 deficiency stimulates necroptosis via Rip3. The effect of antioxidants is not limited to neurodegeneration; it also affects mitochondrial shape.13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar As described above, the loss of Drp1 leads to the formation of large spherical mitochondria after mitochondrial elongation. Mitochondrial elongation directly results from continuous fusion over decreased mitochondrial division, and the second morphological transformation into spheres is mediated by oxidative stress, although its exact molecular mechanisms currently remain unknown.13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar Sphere formation can be suppressed via antioxidant treatment in Drp1-KO Purkinje cells.13Kageyama Y. Zhang Z. Roda R. Fukaya M. Wakabayashi J. Wakabayashi N. Kensler T.W. Reddy P.H. Iijima M. Sesaki H. Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage.J Cell Biol. 2012; 197: 535-551Crossref PubMed Scopus (190) Google Scholar To determine the impact of the loss of Rip3 on mitochondrial morphology in Purkinje cells, cerebellar sections were stained using antibodies to a mitochondrial protein, pyruvate dehydrogenase, and carbonic anhydrase 8. Similar to antioxidants, Rip3 deletion also significantly increased the number of Purkinje neurons that contained nonspherical mitochondria in double-KO mice (Figure 2, A and B). This suppression of sphere formation is not simply because of the increased number of surviving Purkinje neurons, because the percentage of Purkinje neurons with nonspherical mitochondria is significantly increased in double-KO mice (Figure 2C). Therefore, Rip3 appears to mediate the morphological transformation of mitochondria from elongated tubules into large spheres induced by oxidative stress when mitochondrial division is inhibited. In this study, we revealed that post-mitotic Purkinje neurons degenerate via necroptotic cell death in response to the loss of mitochondrial division. Our data suggest that the necroptotic mechanism can both still function in the absence of Drp1 and significantly contribute to neurodegeneration in vivo. Because Drp1-KO Purkinje neurons die in the absence of Rip3, it is important to determine the precise cell-death mechanisms that account for the remaining cell death. Because Purkinje cells undergo necroptosis without Drp1, these cells may also undergo apoptosis in the absence of Drp1. We are planning to address this key question in our future studies. We thank Dr. Vishva M. Dixit (Genentech) for providing us with receptor-interacting protein kinase 3–knockout mice; and past and present members of the Iijima and Sesaki laboratories for helpful discussions and technical assistance. Download .pdf (2.85 MB) Help with pdf files Supplemental Figure S1Purkinje cells (PCs) in double-KO mice at 5 months. A: Double-KO mice were fixed at an age of 5 months. Sagittal sections were cut around the median line of the cerebellum and stained using anti–carbonic anhydrase 8 antibodies. B: Quantification of Purkinje cell density. The number of soma of the Purkinje cells was measured and normalized to the length of the Purkinje cell layer. The same data presented in Figure 1B are shown for 3-month-old mice to compare them with 5-month-old mice. The t-test was used to statistically analyze the difference between 3- and 5-month-old mice. Values represent means ± SEM (B). n = 7 (B, 3 months); n = 3 (B, 5 months). ∗∗∗P < 0.001. Scale bar = 1 mm (A).