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

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.