Reevaluation of neurodegeneration in lurcher mice: constitutive ion fluxes cause cell death with, not by, autophagy.
The lurcher (Lc) mice have served as a valuable model for neurodegeneration for decades. Although the responsible mutation was identified in genes encoding delta2 glutamate receptors (GluD2s), which are predominantly expressed in cerebellar Purkinje cells, how the mutant receptor (GluD2(Lc)) 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 GluD2(Lc) in heterologous cells and cultured neurons, they were blocked by the introduction of mutations in the channel pore domain of GluD2(Lc) or by removal of extracellular Na(+). In addition, although GluD2(Lc) 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 GluD2(Lc) 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 GluD2(Lc)-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.
Nishiyama
Jun
J
Department of Physiology, School of Medicine, Keio University, Tokyo 160-8582, Japan.
Matsuda
Keiko
K
Kakegawa
Wataru
W
Yamada
Nobuaki
N
Motohashi
Junko
J
Mizushima
Noboru
N
Yuzaki
Michisuke
M
D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000502:physiology; D001343:Autophagy; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D002352:Carrier Proteins / Q000502:physiology; D002412:Cations; D016923:Cell Death; D002478:Cells, Cultured; D004789:Enzyme Activation; D000077614:Golgi Matrix Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008818:Mice, Neurologic Mutants; D008869:Microtubule-Associated Proteins / Q000235:genetics; D009410:Nerve Degeneration / Q000378:metabolism / Q000473:pathology*; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D011689:Purkinje Cells / Q000166:cytology / Q000378:metabolism; D017470:Receptors, Glutamate / Q000096:biosynthesis / Q000235:genetics; D012964:Sodium / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C495773:Atg5 protein, mouse; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; C115010:Becn1 protein, mouse; D002352:Carrier Proteins; D002412:Cations; D000077614:Golgi Matrix Proteins; C435484:Gopc protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D017470:Receptors, Glutamate; C107913:glutamate receptor delta 2; D000255:Adenosine Triphosphate; D012964:Sodium; D055372:AMP-Activated Protein Kinases; D002154:Calpain; D002118:Calcium