The answer to the first question is that expression of deltaCAT PS1 is sufficient to rescue the developmental deficits in PS1 KO mice and the production of Aβ peptides in brains of these animals. This is not terribly surprising in view of evidence showing that Notch processing could be rescued in PS1-deficient fibroblasts that ectopically expressed this transgene, and that...  Read more

The answer to the first question is that expression of deltaCAT PS1 is sufficient to rescue the developmental deficits in PS1 KO mice and the production of Aβ peptides in brains of these animals. This is not terribly surprising in view of evidence showing that Notch processing could be rescued in PS1-deficient fibroblasts that ectopically expressed this transgene, and that Aβ production was similar in cells that expressed wildtype versus delCAT PS1 (Saura et al. 2000).

On the other hand, the importance of the aspartate 257 on intramembranous processing of Notch and AβPP is enigmatic. I don't think this paper offers any insight into the issue. At least three reports using transfected mammalian cells have shown that the expression of D257A PS1 blocks processing of Notch but does not effect Aβ production. Indeed, Zheng et al show that when placed on a PS1 knockout background, the transgenes expressing D257A PS1 fail to rescue the developmental deficits in the KO animals. Zheng et al also confirm that expression of the D257A variant in transfected PS1-deficient fibroblasts fails to rescue Notch processing. This has already been published by Haass, Sisodia and others.

But is this relevant in the in vivo paradigm? Certainly, there is rescue by wild-type human PS1 driven by the thy1 promoter, but this is the highest expressing line that was generated in their laboratory. In fact, in adult brain, the level of steady-state expression in the D257A lines is less than a tenth of that seen in the PS1 wildtype line (17-3) or any of the deltaCAT lines (see Fig. 1D). Thus, the failure of the D257A transgenes to rescue the PS1KO phenotype is completely silent. I argue that expression is not high enough in any one of these lines to see any rescue. Considering that in some lines, even substantial levels of expression of deltaCAT (and several wt PS1 lines; Qian et al., 1998) failed to rescue all of the developmental deficits in axial segmentation, one has to question the level at which thy1 transgenes are expressed in embryonic tissue. This is a significant issue when considering the Aβ analysis in Figure 4. There is no data about the level of transgene expression in primary neurons when they are being assayed.

The bottom line is that the alleged influence of the expression of the D257A variant in transgenic mice is silent both with respect to Notch processing and Ab production. In the best-case scenario, transgenic expression of the D257A PS1 variant should have resulted in the generation of a PS1 KO mouse. We now know that ectopic expression of human PS1 (wildtype or FAD-linked mutants in mice) and in transfected cells leads to the steady-state "replacement" of endogenous PS1 and PS2 expression due to competition for limiting cellular factors. In transfected cells, even the D257A and D385A variants replace endogenous PS1. If there was any rescue activity of the D257A variant in the present transgenic experiment by Zheng et al., it would be impossible to detect because the expression levels simply are not high enough. I would bet that those transgenic lines with highest expression of D257A died in utero and that the only survivors are those that express very low levels of transgene product, where there is miniscule "replacement" of endogenous PS1 polypeptides.

View all comments by Sangram Sisodia

Reply by Hui Zheng
Dr. Sisodia challenged our data that hPS1D257A is negative in Notch and AβPP processing and Aβ peptides production in vivo by suggesting it is due to the low levels of expression by the human Thy-1 promoter. We argue against this interpretation for the following reasons:

1) Dr. Sisodia pointed out "In fact, in adult brain, the level of steady-state expression in the D257A lines is less than a tenth of that seen in the PS1 wildtype line (17-3) or any of the deltaCAT lines (see Fig. 1D)." This statement is incorrect because the Western blot was done using embryonic (E14.5), not adult brain. The main point of Fig. 1D is that delatCAT PS1 forms a truncated CTF, whereas only full-length protein can be detected by PS1D257A. It is not a quantitative blot, therefore, "less than a tenth of that seen in¡K" is not valid. Quantitative comparison of multiple transgenic lines shows that expression levels of D257A lines 7 and 4 are comparable to that of deltaCAT lines 3 and 6, respectively (Table 1); both of the latter lines rescue the PS1 null lethality....  Read more

Reply by Hui Zheng
Dr. Sisodia challenged our data that hPS1D257A is negative in Notch and AβPP processing and Aβ peptides production in vivo by suggesting it is due to the low levels of expression by the human Thy-1 promoter. We argue against this interpretation for the following reasons:

1) Dr. Sisodia pointed out "In fact, in adult brain, the level of steady-state expression in the D257A lines is less than a tenth of that seen in the PS1 wildtype line (17-3) or any of the deltaCAT lines (see Fig. 1D)." This statement is incorrect because the Western blot was done using embryonic (E14.5), not adult brain. The main point of Fig. 1D is that delatCAT PS1 forms a truncated CTF, whereas only full-length protein can be detected by PS1D257A. It is not a quantitative blot, therefore, "less than a tenth of that seen in¡K" is not valid. Quantitative comparison of multiple transgenic lines shows that expression levels of D257A lines 7 and 4 are comparable to that of deltaCAT lines 3 and 6, respectively (Table 1); both of the latter lines rescue the PS1 null lethality. Thus, the failure of hPS1D257A to exhibit developmental activity cannot be interpreted as the silence of the transgene.

2) We have performed glycerol gradient analysis of the endogenous and transgenic PS1 proteins and their complexes using E14.5 brains of various genotypes. The result is shown below:

The data clearly indicate this: In contrast to the endogenous mouse PS1 (panel D), wildtype hPS1 (panel A), or deltaCAT PS1 (panel E), where the PS1 proteins are incorporated into high molecular weight (HMW) complexes, D257A protein forms only low molecular weight-complex with or without endogenous mouse PS1 (panels C and B, respectively). Importantly, the D257A protein is expressed to a level similar to that of endogenous PS1 (panels C versus D). Therefore, we suggest that the D257A mutant's failure to form HMW complex is the underlying cause for its inability to process Notch and AβPP, a result previously reported by Peter St. George Hyslop's group (Yu et al., 1998).

3) In the end, Dr. Sisodia stated, "I would bet that those transgenic lines with highest expression of D257A died in utero and that the only survivors are those that express very low levels of transgene product." The lines we analyzed are not "those that express very low levels of transgene product." We generated the deltaCAT and D257A lines in parallel: The detalCAT-positive lines were produced at a frequency of 10.4 percent (7/53), while that of D257A was 20 percent (11/55). Considering that the normal range is 5-20 percent, a 20 percent ratio argues against in utero lethality. I do agree that very high levels of transgene expression that result in the "replacement" of the endogenous protein will lead to in utero lethality, it nevertheless represents an unphysiological state and requires significantly higher transgene expression compared to that of endogenous PS1. It is not the case in our system using the human Thy-1 promoter. Such a high level of expression may lead to non-specific consequences and, therefore, complicate data interpretation (see De Strooper and Annaert, 2001).

View all comments by Hui Zheng

1) Dr. Sisodia pointed out "In fact, in adult brain, the level of steady-state expression in the D257A lines is less than a tenth of that seen in the PS1 wildtype line (17-3) or any of the deltaCAT lines (see Fig. 1D)". This statement is incorrect because the Western blot was done using embryonic (E14.5), not adult brain. The main point of Fig. 1D is that delatCAT PS1 forms a truncated CTF, whereas only full-length protein can be detected by PS1D257A. It is not a quantitative blot, therefore, "less than a tenth of that seen in¡K" is not valid. Quantitative comparison of multiple transgenic lines shows that expression levels of D257A lines 7 and 4 are comparable to that of deltaCAT lines 3 and 6, respectively (Table 1); both of the latter lines rescue the PS1 null lethality. Thus, the...  Read more

1) Dr. Sisodia pointed out "In fact, in adult brain, the level of steady-state expression in the D257A lines is less than a tenth of that seen in the PS1 wildtype line (17-3) or any of the deltaCAT lines (see Fig. 1D)". This statement is incorrect because the Western blot was done using embryonic (E14.5), not adult brain. The main point of Fig. 1D is that delatCAT PS1 forms a truncated CTF, whereas only full-length protein can be detected by PS1D257A. It is not a quantitative blot, therefore, "less than a tenth of that seen in¡K" is not valid. Quantitative comparison of multiple transgenic lines shows that expression levels of D257A lines 7 and 4 are comparable to that of deltaCAT lines 3 and 6, respectively (Table 1); both of the latter lines rescue the PS1 null lethality. Thus, the failure of hPS1D257A to exhibit developmental activity cannot be interpreted as the silence of the transgene.

2) We have performed glycerol gradient analysis of the endogenous and transgenic PS1 proteins and their complexes using E14.5 brains of various genotypes. The result is shown below:

The data clearly indicate this: In contrast to the endogenous mouse PS1 (panel D), wildtype hPS1 (panel A), or deltaCAT PS1 (panel E), where the PS1 proteins are incorporated into high molecular weight (HMW) complexes, D257A protein forms only low molecular weight-complex with or without endogenous mouse PS1 (panels C and B, respectively). Importantly, the D257A protein is expressed to a level similar to that of endogenous PS1 (panels C versus D). Therefore, we suggest that the D257A mutant's failure to form HMW complex is the underlying cause for its inability to process Notch and AβPP, a result previously reported by Peter St. George Hyslop's group (Yu et al., 1998).

3) In the end, Dr. Sisodia stated, "I would bet that those transgenic lines with highest expression of D257A died in utero and that the only survivors are those that express very low levels of transgene product". The lines we analyzed are not "those that express very low levels of transgene product". We generated the deltaCAT and D257A lines in parallel: The detalCAT-positive lines were produced at a frequency of 10.4 percent (7/53), while that of D257A was 20 percent (11/55). Considering that the normal range is 5-20 percent, a 20 percent ratio argues against in utero lethality. I do agree that very high levels of transgene expression that result in the "replacement" of the endogenous protein will lead to in utero lethality, it nevertheless represents an unphysiological state and requires significantly higher transgene expression compared to that of endogenous PS1. It is not the case in our system using the human Thy-1 promoter. Such a high level of expression may lead to non-specific consequences and, therefore, complicate data interpretation (see De Strooper and Annaert, 2001).

View all comments by Hui Zheng