A paper in the current PNAS is advancing the controversy over the identity of the catalytic subunit of γ-secretase, the protease responsible for the cleavage of AβPP and the generation of Aβ variants Aβ40 and Aβ42. While the evidence is inconclusive, many researchers consider presenilin-1 (PS1), mutations of which cause familial Alzheimer's disease, to be the prime candidate for this catalytic role (see related news item). Detractors point to numerous inconsistencies in the data, including that PS1 and the γ-secretase proteolytic activity are localized to different cellular compartments, and the finding that overexpression of inactive, mutant PS1 fails to affect production of Aβ. Proponents point to the presence of endogenous active PS1 as a complicating factor in these transfection experiments.

This latter issue is now being addressed by Hui Zheng, Baylor College of Medicine, Houston, with colleagues at RIKEN and Gunma University, Japan, and elsewhere. The authors have expressed mutant human PS1 in presenilin knockout mice, eliminating any contribution by endogenous protein. Their work appears in next week's PNAS and is currently available online.

Xuefeng Xia et al. found that mutating one of the proposed essential aspartic acid residues of PS1 to alanine (D257A) has profound consequences. D257A mutants failed to rescue PS knockout mice, which die shortly before or after birth due to developmental defects. Cultured primary neurons from these animals failed to process AβPP as judged by the profound decrease in Aβ40 and Aβ42 production. Normal development and AβPP processing was rescued, however, when the authors instead used a presenilin construct that misses amino acids 340-371, the binding loop for one putative binding partner of PS1, β-catenin. This experiment suggests not only that PS1 is essential for γ-secretase activity, but that normal AβPP processing can proceed in the absence of any interaction with β-catenin.—Tom Fagan

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  1. The manuscript by Zheng et al. reports that the aspartate aa 257 in transmembrane domain six is indispensable for Notch processing and Aβ production. The authors also tested the possibility that expression of a thy1 promoter-driven PS1 transgene encoding PS1 deleted for a domain that is a binding site for β-catenin (deltaCAT) might rescue the developmental deficits in PS1 knockout mice and/or affect Aβ production. More importantly, the authors asked whether a thy1 promoter-driven transgene encoding the PS1 D257A variant could rescue the developmental deficits in PS1 KO mice and whether Aβ production could be rescued in primary neurons obtained from embryos that have the transgene placed on a KO background.

    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.

    References:

    . The nonconserved hydrophilic loop domain of presenilin (PS) is not required for PS endoproteolysis or enhanced abeta 42 production mediated by familial early onset Alzheimer's disease-linked PS variants. J Biol Chem. 2000 Jun 2;275(22):17136-42. PubMed.

    . Mutant human presenilin 1 protects presenilin 1 null mouse against embryonic lethality and elevates Abeta1-42/43 expression. Neuron. 1998 Mar;20(3):611-7. PubMed.

  2. 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:

    image

    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).

    References:

    . The presenilin 1 protein is a component of a high molecular weight intracellular complex that contains beta-catenin. J Biol Chem. 1998 Jun 26;273(26):16470-5. PubMed.

    . Presenilins and the intramembrane proteolysis of proteins: facts and fiction. Nat Cell Biol. 2001 Oct;3(10):E221-5. PubMed.

  3. 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:

    image

    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).

    References:

    . The presenilin 1 protein is a component of a high molecular weight intracellular complex that contains beta-catenin. J Biol Chem. 1998 Jun 26;273(26):16470-5. PubMed.

References

News Citations

  1. Presenilin—Guilty of Proteolysis by Association?

Further Reading

Primary Papers

  1. . The aspartate-257 of presenilin 1 is indispensable for mouse development and production of beta-amyloid peptides through beta-catenin-independent mechanisms. Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8760-5. PubMed.