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Articles, Neurobiology of Disease

Reevaluation of Neurodegeneration in lurcher Mice: Constitutive Ion Fluxes Cause Cell Death with, Not by, Autophagy

Jun Nishiyama, Keiko Matsuda, Wataru Kakegawa, Nobuaki Yamada, Junko Motohashi, Noboru Mizushima and Michisuke Yuzaki
Journal of Neuroscience 10 February 2010, 30 (6) 2177-2187; https://doi.org/10.1523/JNEUROSCI.6030-09.2010
Jun Nishiyama
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Keiko Matsuda
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Wataru Kakegawa
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Nobuaki Yamada
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Junko Motohashi
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Noboru Mizushima
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Michisuke Yuzaki
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Abstract

The lurcher (Lc) mice have served as a valuable model for neurodegeneration for decades. Although the responsible mutation was identified in genes encoding δ2 glutamate receptors (GluD2s), which are predominantly expressed in cerebellar Purkinje cells, how the mutant receptor (GluD2Lc) triggers cell death has remained elusive. Here, taking advantage of recent knowledge about the domain structure of GluD2, we reinvestigated Lc-mediated cell death, focusing on the “autophagic cell death” hypothesis. Although autophagy and cell death were induced by the expression of GluD2Lc in heterologous cells and cultured neurons, they were blocked by the introduction of mutations in the channel pore domain of GluD2Lc or by removal of extracellular Na+. In addition, although GluD2Lc is reported to directly activate autophagy, mutant channels that are not associated with n-PIST (neuronal isoform of protein-interacting specifically with TC10)–Beclin1 still caused autophagy and cell death. Furthermore, cells expressing GluD2Lc showed decreased ATP levels and increased AMP-activated protein kinase (AMPK) activities in a manner dependent on extracellular Na+. Thus, constitutive currents were likely necessary and sufficient to induce autophagy via AMPK activation, regardless of the n-PIST–Beclin1 pathway in vitro. Interestingly, the expression of dominant-negative AMPK suppressed GluD2Lc-induced autophagy but did not prevent cell death in heterologous cells. Similarly, the disruption of Atg5, a gene crucial for autophagy, did not prevent but rather aggravated Purkinje-cell death in Lc mice. Furthermore, calpains were specifically activated in Lc Purkinje cells. Together, these results suggest that Lc-mediated cell death was not caused by autophagy but necrosis with autophagic features both in vivo and in vitro.

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The Journal of Neuroscience: 30 (6)
Journal of Neuroscience
Vol. 30, Issue 6
10 Feb 2010
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Reevaluation of Neurodegeneration in lurcher Mice: Constitutive Ion Fluxes Cause Cell Death with, Not by, Autophagy
Jun Nishiyama, Keiko Matsuda, Wataru Kakegawa, Nobuaki Yamada, Junko Motohashi, Noboru Mizushima, Michisuke Yuzaki
Journal of Neuroscience 10 February 2010, 30 (6) 2177-2187; DOI: 10.1523/JNEUROSCI.6030-09.2010

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Reevaluation of Neurodegeneration in lurcher Mice: Constitutive Ion Fluxes Cause Cell Death with, Not by, Autophagy
Jun Nishiyama, Keiko Matsuda, Wataru Kakegawa, Nobuaki Yamada, Junko Motohashi, Noboru Mizushima, Michisuke Yuzaki
Journal of Neuroscience 10 February 2010, 30 (6) 2177-2187; DOI: 10.1523/JNEUROSCI.6030-09.2010
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  • Aggravation of Lc-induced cell death in Atg5-null Purkinje cells
    Michisuke Yuzaki
    Published on: 16 March 2010
  • Autophagy in Lurcher mice: an unsolved �death� question
    Zhenyu Yue
    Published on: 01 March 2010
  • Published on: (16 March 2010)
    Page navigation anchor for Aggravation of Lc-induced cell death in Atg5-null Purkinje cells
    Aggravation of Lc-induced cell death in Atg5-null Purkinje cells
    • Michisuke Yuzaki, Professor
    • Other Contributors:
      • Jun Nishiyama

    In their eLetter, Yue cast some doubt on the main conclusion drawn in our study that Purkinje cell degeneration in lurcher (Lc) mice is not caused by autophagy, based on two arguments.

    To examine the role of autophagy in Lc-mediated cell death in vivo, we crossed Lc mice with conditional Atg5 knockout mice (Atg5f/f; pcp2-Cre), in which Atg5, a gene indispensable for autopha...

    Show More

    In their eLetter, Yue cast some doubt on the main conclusion drawn in our study that Purkinje cell degeneration in lurcher (Lc) mice is not caused by autophagy, based on two arguments.

    To examine the role of autophagy in Lc-mediated cell death in vivo, we crossed Lc mice with conditional Atg5 knockout mice (Atg5f/f; pcp2-Cre), in which Atg5, a gene indispensable for autophagy, was selectively excised in cerebellar Purkinje cells. Yue wrote that it was unclear whether the Atg5 protein had been absent before the Lc-induced cell death started. Indeed, we previously showed that Cre-mediated deletion of the Atg5 gene was observed as early as P6 and was fully established by P21 in these mice (Nishiyama et al., 2007). Thus, we are aware that the Atg5 gene and its encoded protein were partially deleted when Lc-mediated cell death started during the second postnatal week.

    To knockout the Atg5 gene much earlier, we also crossed Lc mice with Atg5f/f; nestin-Cre mice, in which the Atg5 protein was deleted by embryonic day 15.5 (Hara et al, 2006). Although we found that more Purkinje cells were degenerated in Lc/+; Atg5f/f; nestin-Cre mice, it was difficult to determine whether Lc-induced cell death was directly affected by the autophagic pathway because Atg5f/f; nestin-Cre mice, without crossing with Lc mice, exhibit progressive neurodegeneration starting a few weeks after birth (Hara et al, 2006). Therefore, we used Atg5f/f; Pcp2-Cre mice, in which Purkinje cells remain normal until 8 weeks after birth (Nishiyama et al., 2007).

    We believe that the most important finding is that although the deletion of the Atg5 protein was partial, Lc-induced Purkinje cell death was not rescued, but was rather aggravated in Atg5f/f; Pcp2-Cre mice, indicating that Lc-mediated cell death was at least not directly caused by autophagy activation in vivo.

    Second, Yue stated that "a cell culture study in which transfection of the delta2 glutamate receptor (GluD2) with the Lc mutation was performed with the knock-down of the essential autophagy gene would have been helpful to assess the role of autophagy." Indeed, we showed that GluD2Lc-mediated cell death was not rescued by expression of a dominant-negative form of AMPK, which completely inhibited the induction of autophagy (Nishiyama et al, 2010, Fig. 5). In addition, overexpression of GluD2Lc killed Atg5-null, as well as wild-type, MEF cells (data not shown). Therefore, the blockade of autophagy did not prevent Lc-induced cell death in vitro.

    Whether the excessive activation of autophagy can kill cells has been a much-debated question, and Lc mutant mice have been cited as an example of cell death resulting from autophagy in vivo.  We believe that our study clearly indicated that Lc-mediated cell death was not caused by autophagy but necrosis with autophagic features both in vivo and in vitro.

    REFERENCES

    Nishiyama J, Miura E, Mizushima N, Watanabe M, Yuzaki M. Aberrant membranes and double-membrane structures accumulate in the axons of Atg5-null Purkinje cells before neuronal death. Autophagy. 2007 Nov-Dec;3(6):591-6.

    Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, Yokoyama M, Mishima K, Saito I, Okano H, Mizushima N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature. 2006 Jun 15;441(7095):885-9.

    Show Less
    Competing Interests: None declared.
  • Published on: (1 March 2010)
    Page navigation anchor for Autophagy in Lurcher mice: an unsolved �death� question
    Autophagy in Lurcher mice: an unsolved �death� question
    • Zhenyu Yue, Associate Professor of Neurology and Neuroscience

    This study has revisited an autophagy-neurodegeneration model in Lurcher (Lc) mice and promoted further discussion regarding the “autophagic cell death” hypothesis. While the study confirmed the previous report by Yue et al (2002) that GluRD2Lc induced autophagy in cultured cells as well as in mice, it also suggested that GluRD2Lc-mediated autophagy and cell death occur via pathways outside the nPIST-Beclin 1 pathway based...

    Show More

    This study has revisited an autophagy-neurodegeneration model in Lurcher (Lc) mice and promoted further discussion regarding the “autophagic cell death” hypothesis. While the study confirmed the previous report by Yue et al (2002) that GluRD2Lc induced autophagy in cultured cells as well as in mice, it also suggested that GluRD2Lc-mediated autophagy and cell death occur via pathways outside the nPIST-Beclin 1 pathway based largely on in vitro studies. The authors showed that GluRD2Lc-induced degeneration is associated with energy crisis and aberrant AMPK activity. This result is interesting but not surprising, considering that constitutive ion influx caused by Lc mutation is expected to cause activation of multiple cellular pathways or responses.

    A critical component of the study is the investigation of the role for autophagy in Purkinje cell death in the animal model (Figure 7). The authors concluded that Lc-mediated cell death is not caused by autophagy, but only has autophagic features. They generated Lc/+; Atg5F/F; Pcp2-Cre mice, in which an essential autophagy gene, Atg5, is supposed to be deleted in Lc Purkinje cells, with the hope of examining how loss of Atg5 affects the process of Purkinje cell death. However, a critical question emerges regarding the timing of loss of the Atg5 gene and protein relative to the degeneration of Purkinje cells. It is known that Lc Purkinje cells start to degenerate at P6-P8, and most die within the second postnatal week. Although Pcp2 promoter turns on in at least a subset of Purkinje cells before birth, the excision of a target gene by Cre recombinase could be delayed and should be carefully examined. Interestingly, a previous study by Nishiyama et al. reported that only a small portion of Purkinje cells have Atg5 excised by P14 (2007), a stage when most Purkinje cells have already degenerated in Lc mice. Furthermore, a complete turnover of Atg5 mRNA and proteins may not occur until a significant period of time after the deletion of Atg5 alleles, resulting in a delay of autophagy ablation. Therefore, it is important to show when Atg5 protein is actually abolished in order to reach a valid conclusion. Unfortunately, this critical piece of evidence has not been provided in this article. As a matter of fact, Komatsu et al. (2007) have previously performed a similar study in Atg7F/F; Pcp2-Cre mice. By performing immunohistochemistry with an anti-Atg7 antibody, these authors showed that Atg7 protein persisted throughout P15 but diminished at P19 in Purkinje cells (Komatsu et al., 2007). This evidence strongly suggests that loss of Atg5 protein in most, if not all, Purkinje cells occurs after P14-15, and one can infer that in Lc/+; Atg5F/F; Pcp2-Cre mice, most Purkinje cells may have already degenerated (by P14) due to the Lc mutation without a chance for the Atg5 deletion to exert its effects. Finally, a cell culture study in which transfection of GluRD2Lc was performed with the knock-down of the essential autophagy gene would have been helpful to assess the role of autophagy. Again, this study is missing. It thus raises doubt on the main conclusion drawn in this study that Lc Purkinje cell degeneration is not caused by autophagy.

    This report by Nishiyama et al. has made a major effort to solve an important question regarding autophagy and cell death. However, given the current report, whether or not autophagy plays an active role in Lc Purkinje cell degeneration and how it is involved still remains unsolved. To answer this question, future experiments will need to recruit novel genetic mouse models that can precisely control the excision of an essential autophagy gene at the appropriate stage of Purkinje cell development.

    Reference:

    Komatsu M, Wang QJ, Holstein GR, Friedrich VL, Jr., Iwata J, Kominami E, Chait, BT, Tanaka K, and Yue Z. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci U S A. 2007;104(36):14489-94.

    Nishiyama J, Miura E, Mizushima N, Watanabe M, Yuzaki M. Aberrant membranes and double-membrane structures accumulate in the axons of Atg5- null Purkinje cells before neuronal death. Autophagy. 2007;3(6):591-6.

    Yue Z, Horton A, Bravin M, DeJager PL, Selimi F, Heintz N. A novel protein complex linking the delta 2 glutamate receptor and autophagy: implications for neurodegeneration in lurcher mice. Neuron. 2002;35(5):921 -33.

    Show Less
    Competing Interests: None declared.

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