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Calpain activation in Alzheimer's disease: Comments on Saito et al.
Submit responseSaito and colleagues stated that 60% of the phenotypes observed in traditional AD mouse models are merely "artifact", based on the absence of these phenotypes in their APP knock-in mouse models carrying two or three distinct APP mutations (Saito et al., 2014). In this line, they claimed that calpain activation, p25 production and Nav1.1 reduction are all artifacts of APP and PS1 overexpression. Careful examination of the limited data presented, however, shows that this conclusion is not strongly supported. The data suffer from absence of some controls and insufficient analysis. Specific points that should be considered are highlighted below:
1. APP duplication is known to cause familial Alzheimer's disease. Similarly, individuals with Down's syndrome caused by trisomy of chromosome 21 (which contains the APP gene) also develop early-onset Alzheimer's disease. Moreover, there is evidence that individual neurons in sporadic AD patients show differences in APP gene copy number (Bushman et al., 2015). Therefore, given that APP duplication (and its overexpression) is a cause of Alzheimer's disease, it is misleading to characterize the increased calpain activity as an artifact of APP overexpression.
2. The authors suggest that the differences they observe between their APP mutant knock-in model and other transgenic models are simply due to the presence or absence of APP overexpression. This is a very limited interpretation. It is well established that AD-related pathogenic phenotypes in mouse models not only relate to APP expression and total Abeta generation, but also to Abeta oligomer formation and localization, both neuroanatomic and subcellular. These important factors have not been analyzed in this paper or the preceding report (Saito et al., 2014).
3. Increased calpain activity and marked reduction of calpastatin (Cast) have not only been shown in mouse models of Alzheimer's disease, but have also been widely reported in post-mortem brain samples of Alzheimer's disease patients (for instance, Saito et al., 1993; Rao et al., 2008; Laske et al., 2015; Kurbatskava et al., 2016). Thus, the lack of increased calpain activity in the mouse model used by Saito and colleagues calls into question the physiological relevance of their model, and whether the APP23 and other transgenic models are closer approximations of the human disease.
4. Increased p25 generation in Cast-KO mice under pathological conditions has been shown previously (Sato et al., 2011). However, in Fig. 1, Cast-KO/APPNL-F/NL-F mice do not show increased p25 signal, which should occur if as expected, it reflects calpain-dependent p35 cleavage. This raises the question of specificity and whether the authors are in fact measuring p25. The authors extensively discuss crosses with a Cast-KO mouse, but they do not show any p25 data from Cast-KO mouse brain or APP23 crossed to Cast-KO. These blots should show elevated p25 signal and would serve as positive controls.
5. The knock-in APPNL-F/NL-F mice were examined only at 2 years of age. It is possible that calpain activity is downregulated by this late age. It is also possible that calpain gene(s) are disrupted in their model. These possibilities may explain why the cross of APPNL-F/NL-F with Cast-KO did not increase p25 generation. Moreover, examination of the gel in Fig. 1 suggests that p35 may be elevated in APPNL-F/NL-F compared with the other genotypes, consistent with calpain downregulation. These control experiments should be conducted before making conclusions. To rigorously assess p25 generation, a broader range of ages should also be analyzed.
A similar concern applies to Fig 2, in which conclusions about sodium channel expression are based solely on a 2-year-old brain sample, and is generalized to suggest that a previous report of sodium channel downregulation in the J20 model was unique to that model. Furthermore, the present paper utilizes Western blotting of whole brain homogenates, a highly insensitive approach for assessing sodium channel regulation in specific interneuron subpopulations. In particular, there is no in situ analysis by immunofluorescence, electrophysiology or other methods.
6. The authors state that calpain-mediated p25 generation is an artifact caused by membrane protein overexpression such as APP or PS1, but they never address this directly. There are multiple studies demonstrating that in the absence of APP overexpression, organotypic hippocampal slices, cultured primary neurons, and even primary microglia exposed to Abeta show increased p25 generation (for instance, Zheng et al., 2005; Ma et al., 2013; Seo et al., 2014). Moreover, PS1 mutations were shown indisputably to disrupt calcium homeostasis and activate calcium-dependent enzymes (Morgan et al., 2007; Muller et al., 2011), which can increase calpain activity and subsequent p25 generation. Extending claims of artifact to PS1-related mouse models when their analysis is limited to APP models is scientifically unwarranted.
7. Finally, there is a large body of literature from many mouse models of neurological disorders that shows p25 production that is unrelated to overexpression of membrane proteins. These include models of tauopathies (Rao et al., 2014), ischemia (Meyer et al., 2014), and Parkinson's disease (Smith et al., 2006). Saito et al (2016) did not cite any of these studies. Furthermore, the statement that "overexpression of a mutant tau protein, a cytosolic protein, exhibited no effect on p25 levels (data not shown)" directly contradicts previous literature, a discrepancy that was not discussed. Based on points 6 and 7, it is misleading to characterize the p25 generation as an artifact of membrane protein overexpression.
References
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Conflict of Interest:
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