Seminal studies of LeBlanc (1995) have shown that primary neurons committed to undergo apoptosis by serum-deprivation produced increased amount of Abeta. These findings have been confirmed in cerebellar granule cells (Galli et al., 1998), in retinoic acid-differentiated neuronal NT2 cells (Gervais et al., 1999) and in chick embryo motoneurons (Barnes et al.,1998). Caspase inhibition has been shown to reduce the effect of apoptosis on Abeta levels (LeBlanc, 1999 ; Gervais et al., 1999).

More recently it has been shown that APP is also a substrate for caspase cleavage (Barnes et al., 1998; Gervais et al., 1999; LeBlanc et al., 1999; Pellegrini et al., 1999; Weidemann et al., 1999). Many recombinant caspases have been shown to cleave APP in an in vitro assay and a major caspase site has been identified at D720 (VEVD). Each of the four caspases 3, 6, 7, and 8 are able to cleave APP at D720 in vitro resulting in the release of a fragment containing the last 31 amino-acids of APP (C31) and in the production of APPdeltaC31 (lacking Ala721 to Asn751). APP caspase-mediated cleavage at...  Read more

Seminal studies of LeBlanc (1995) have shown that primary neurons committed to undergo apoptosis by serum-deprivation produced increased amount of Abeta. These findings have been confirmed in cerebellar granule cells (Galli et al., 1998), in retinoic acid-differentiated neuronal NT2 cells (Gervais et al., 1999) and in chick embryo motoneurons (Barnes et al.,1998). Caspase inhibition has been shown to reduce the effect of apoptosis on Abeta levels (LeBlanc, 1999 ; Gervais et al., 1999).

More recently it has been shown that APP is also a substrate for caspase cleavage (Barnes et al., 1998; Gervais et al., 1999; LeBlanc et al., 1999; Pellegrini et al., 1999; Weidemann et al., 1999). Many recombinant caspases have been shown to cleave APP in an in vitro assay and a major caspase site has been identified at D720 (VEVD). Each of the four caspases 3, 6, 7, and 8 are able to cleave APP at D720 in vitro resulting in the release of a fragment containing the last 31 amino-acids of APP (C31) and in the production of APPdeltaC31 (lacking Ala721 to Asn751). APP caspase-mediated cleavage at D720 has been shown to occur in vivo after induction of brain ischemic injury and in brains of human AD subjects (Gervais et al., 1999; Lu et al., 2000). These findings raise the possibility that caspase-mediated cleavage of APP mediates the increased Abeta production during apoptosis. Gervais et al. (1999) have reported that the overexpression of APPdeltaC31, corresponding to the product of caspase proteolysis at D720, resulted in a 5 fold increase in total Abeta production in rat B103 cells. However, caspase cleavage at D720 of APP is expected to remove the internalization motif (YENP), present at the C-terminus of APP that is responsible for clathrin-mediated internalization of APP and the lack of APP internalization has been shown to decrease Abeta generation in CHO cells (Perez et al., 1999).

In a paper in press in JBC, published on June 7, 2001 as Manuscript M102456200, Soriano et al. reported that caspase-mediated cleavage of APP at its cytosolic tail (D720) is not amyloidogenic and that induction of apoptosis by serum withdrawal in B103 cells did not increase Abeta generation. The authors showed that APP endocytosis and Abeta generation was impaired in both CHO and B103 cell lines overexpressing APPdeltaC31 as well as in cells that overexpress APPdeltaC due to the absence of the YENP motif. These data are in contrast with Gervais et al. 1999 who reported a 5-fold increase in Abeta production when APPdeltaC31 was overexpressed in B103 cells. Soriano et al. explained the different results suggesting that Gervais et al. actually measured an increase in p3 using for their ELISA assay an antibody that recognize both Abeta and p3. The authors actually showed that p3 production was increased in cells overexpressing both APPdeltaC31 and APPdeltaC.

In addition, Soriano et al. reported that Abeta generation was not increased when B103 cells were serum deprived for 18 hrs. However the authors did not report data regarding the percentage of viable cells at the time point when Abeta was measured. Since Abeta production directly correlates with the number of viable cells we will expect that a decrease in viability would either lead to a decrease in Abeta production or mask the increase in Abeta generation associated with apoptosis. On the other hand, since the expression and/or compartmentalization of caspases is cell-type dependent (for review see Earnshaw et al., 1999), the authors suggested that the absence of the effect on Abeta generation after serum deprivation in B103 cells could depend on the cell line or/and the apoptotic stressor used.

The authors concluded that the amyloidogenic pathway of the beta-amyloid precursor protein is independent of its cleavage by caspases. However, since other caspase sites in APP have been recently identified, at the N-terminal domain of APP D197 and D219 (Gervais et al., 1999), at D554 (Barnes et al., 1998), at D653 in the beta-secretase region (Gervais et al., 1999; LeBlanc et al., 1999), the data reported by Soriano et al. seem only to exclude that caspase cleavage at D720 is implicated in the amyloidogenic pathway. It possible that caspase-mediated cleavage at sites different from D720 might be implicated in increased Abeta generation during apoptosis.

References

Barnes NY, Li L, Yoshikawa K, Schwartz LM, Oppenheim RW, Milligan CE. Increased production of amyloid precursor protein provides a substrate for caspase-3 in dying motoneurons. J Neurosci 1998;18(15):5869-80; Abstract.

Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 1999;68:383-424; Abstract.

Galli C, Piccini A, Ciotti MT, Castellani L, Calissano P, Zaccheo D, Tabaton M. Increased amyloidogenic secretion in cerebellar granule cells undergoing apoptosis. Proc Natl Acad Sci U S A 1998;95(3):1247-52; Abstract.

Gervais FG, Xu D, Robertson GS, Vaillancourt JP, Zhu Y, Huang J, LeBlanc A, Smith D, Rigby M, Shearman MS, et al. Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid- beta precursor protein and amyloidogenic A beta peptide formation. Cell 1999;97(3):395-406; Abstract.

LeBlanc A. Increased production of 4 kDa amyloid beta peptide in serum deprived human primary neuron cultures: possible involvement of apoptosis. J Neurosci 1995;15(12):7837-46; Abstract.

LeBlanc A, Liu H, Goodyer C, Bergeron C, Hammond J. Caspase-6 role in apoptosis of human neurons, amyloidogenesis, and Alzheimer's disease. J Biol Chem 1999;274(33):23426-36; Abstract.

Lu DC, Rabizadeh S, Chandra S, Shayya RF, Ellerby LM, Ye X, Salvesen GS, Koo EH, Bredesen DE. A second cytotoxic proteolytic peptide derived from amyloid beta- protein precursor [see comments]. Nat Med 2000;6(4):397-404; Abstract.

Pellegrini L, Passer BJ, Tabaton M, Ganjei JK, D'Adamio L. Alternative, non-secretase processing of Alzheimer's beta-amyloid precursor protein during apoptosis by caspase-6 and -8. J Biol Chem 1999;274(30):21011-6; Abstract.

Perez RG, Soriano S, Hayes JD, Ostaszewski B, Xia W, Selkoe DJ, Chen X, Stokin GB, Koo EH. Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42. J Biol Chem 1999;274(27):18851-6; Abstract.

Weidemann A, Paliga K, U Dr, Reinhard FB, Schuckert O, Evin G, Masters CL. Proteolytic processing of the Alzheimer's disease amyloid precursor protein within its cytoplasmic domain by caspase-like proteases. J Biol Chem 1999;274(9):5823-9; Abstract.

View all comments by Rudy Tanzi
View all comments by Giuseppina Tesco

"This provides important information relevant to a continuing debate over the mechanisms underlying production of Ab."

View all comments by Benjamin Wolozin

The point of our paper was the surprising result from Gervais and colleagues that caspase cleavage of APP resulted in more, rather than less, A-beta. The five fold increase in A-beta was striking, making it comparable to the effects of the APP "Swedish" mutation. The authors built a nice hypothesis that caspase cleavage results in more A-beta, then more A-beta toxicity, more caspase cleavage, and so on, leading to an escalating cycle that kills neurons in Alzheimer's disease. The problem is that it did not make sense from the standpoint of current knowledge of A-beta production through the endocytic pathway, a step that requires an intact APP c-terminal tail. Our results did confirm our expectation that caspase cleavage lead to a loss rather than an increase in A-beta. We were careful to use not only our usual CHO cell line but also the B103 cells used by Gervais and colleagues, eliminating the variable of cell line differences. However, we really couldn't explain their results other than it not fitting current model of A-beta generation. The review does correctly point out...  Read more

The point of our paper was the surprising result from Gervais and colleagues that caspase cleavage of APP resulted in more, rather than less, A-beta. The five fold increase in A-beta was striking, making it comparable to the effects of the APP "Swedish" mutation. The authors built a nice hypothesis that caspase cleavage results in more A-beta, then more A-beta toxicity, more caspase cleavage, and so on, leading to an escalating cycle that kills neurons in Alzheimer's disease. The problem is that it did not make sense from the standpoint of current knowledge of A-beta production through the endocytic pathway, a step that requires an intact APP c-terminal tail. Our results did confirm our expectation that caspase cleavage lead to a loss rather than an increase in A-beta. We were careful to use not only our usual CHO cell line but also the B103 cells used by Gervais and colleagues, eliminating the variable of cell line differences. However, we really couldn't explain their results other than it not fitting current model of A-beta generation. The review does correctly point out that our study addressed only caspase cleavage in the C-terminal tail. We did not address the other cleavage sites although as we and Rudy (in a previous review) have pointed out, it is unlikely that cleavage at the N-terminus of A-beta is by caspases either, unless the N-terminus is truncated by one residue. Finally, the serum withdrawal experiment was meant to show that a cellular insult can result in caspase cleavage of APP. We did not want to delve into the issue of A-beta generation during apoptosis. This, as pointed out by Giuseppina and Rudy, activates a whole hosts of cellular changes, including activation of caspases, that complicate the picture significantly.

View all comments by Eddie Koo