Cover Image

Not for Sale



View/Hide Left Panel

FIG. 1. Aβ is generated from precursor protein, APP. N, N terminus; C, C terminus.

mer’s disease, perhaps as a consequence of loss of cholinergic stimulation. The “uncleaved” APP could then be cleaved by aberrant proteolytic events, perhaps mediated by lysosomal enzymes, generating Aβ.

sAPP Production: β-Secretase. The first piece of evidence that Aβ production may not be aberrant after all was provided by the observation that both APP-transfected HEK293 cells (13) as well as fetal neuronal cultures (14) constitutively release Aβ 1–40 into the culture medium, i.e., Aβ generation and extracellular release are by-products of normal cellular metabolism of APP. This conclusion, dramatic at the time, has since been confirmed by many investigators and has come to be widely accepted.

Shortly thereafter, it was shown that a truncated form of sAPP was released from HEK293 cells transfected with APP, as well as from primary fetal human neuronal cultures (15). Using a neoepitope-specific antibody, these investigators showed that the truncated sAPP ended precisely at Met-596, a marker of specific endoproteolytic cleavage immediately N-terminal to the Aβ sequence. This β-sAPP made up a much larger proportion of total sAPP in the neuronal culture CM than in the HEK293 cell CM, suggesting that this alternative secretory cleavage, by the so-called β-secretase, was more prominent in cells derived from the central nervous system.

The consequences of these two pivotal observations were that it became possible to measure three key metabolites of APP (α-sAPP, β-sAPP, and Aβ) in a cellular context and especially to look for both inhibitors and potential stimulators of Aβ release under defined conditions.

Stimulated Release of sAPP: Effect on Aβ. Phorbol esters, such as phorbol 12-myristate 13-acetate or phorbol dibutyrate, have been used widely to stimulate sAPP release in a variety of cellular systems. Early results suggested that stimulation of sAPP release was accompanied, reciprocally, by a decrease in Aβ release (16). However, subsequent analysis in a neuroblastoma cell line in culture showed that stimulated release of sAPP was not always accompanied by decreased Aβ (17). Although phorbol 12-myristate 13-acetate virtually universally stimulates α-sAPP production, there is little, if any, effect on β-sAPP levels, and the reduction of Aβ is often only transient (J.Knops and S.S., unpublished observations). No effect on synthesis of APP was seen in these experiments. Thus, there is not necessarily a mutually exclusive relationship between α-and β-secretory cleavages, a conclusion that has become more apparent as other pharmacological agents for affecting APP metabolism have become available.

Bafilomycin and β-sAPP Inhibition. A double mutation of codons 670/671 of APP, replacing the Lys-Met sequence with Asn-Leu (18) and segregating with very early-onset Alzheimer’s disease with classic pathologic hallmarks, was described in 1992. Transfection of HEK293 cells with cDNA constructs coding for the mutated protein led to a 6-fold increase in extracellularly released Aβ (19) compared with wild-type (Wt) APP. Concurrent analysis of the sAPP species released showed that there was also a substantial increase in the β-sAPP being released from such cells. The so-called Swedish mutation in APP thus seems to exert its pathogenic effect via an increased production of Aβ, mediated by increased β-secretase cleavage in the mutated protein. This observation provided, not only a mechanistic explanation for a pathogenic mutation, but also a cellular system, relevant to the underlying disease model, in which to study pharmacological agents that can selectively inhibit the formation of Aβ.

A specific and potent inhibitor of vacuolar ATPases, bafilomycin, was shown to inhibit β-sAPP selectively, but not α-sAPP, both from HEK293 cells transfected with APP Swedish mutants and from fetal neuronal cultures (20). This effect was ascribed to the known pharmacological activity of bafilomycin, treatment with which leads to the elevation of intravesicular pH in a variety of acidic organelles, including, but not restricted to, endosomes and lysosomes (21). The concordance of the data obtained from studies with both the mutant APP-transfected cells and fetal neuronal cultures metabolizing endogenous Wt APP showed (i) that selective inhibition of β-secretase cleavage results in inhibition of Aβ release and (ii) that α-sAPP release is not affected under these conditions. Further, these data provided indirect but convincing evidence that acidic intracellular conditions are most conducive to efficient β-secretase processing of APP.

Like APP, a number of other membrane-bound proteins are “shed” from the cell surface, often in response to stimulation by phorbol esters (22). A pathologically important protein in this regard is pro-tumor necrosis factor-α (proTNF-α), which undergoes cell-surface proteolysis by an “α-secretase-like” enzyme to release circulating TNF. The purification and identification of the TNF-α-converting enzyme (TACE) as a membrane-bound metalloprotease (23) led to speculation that, like TACE, APP α-secretase is also a member of the adamalysin protease family. Cells deficient in TACE do not show any defect in constitutive α-cleavage of APP (24); however, no stimulated release of sAPP is evident on treatment with phorbol esters, suggesting that TACE plays a key role in regulated, but not constitutive, α-cleavage of APP. Metalloprotease inhibitors directed toward such proteases inhibit α-sAPP release from Chinese hamster ovary (CHO) cells in a dose-dependent manner (25), but such treatments have no significant effect on either β-sAPP or Aβ (E.Goldbach, S. Suomensaari, J.Knops, and S.S., unpublished observations).

The results of the phorbol ester, bafilomycin, and metalloprotease inhibitor studies strongly suggest that a simple reciprocal relationship does not exist between α- and β-cleavage or between sAPP production and Aβ release. It seems most likely that α-secretase and β-secretase are cellularly segregated, mechanistically distinct enzymes, and it is the direct action of the latter that correlates most with Aβ release.

Pathogenic Mutations in APP. Three separate missense mutations in APP, occurring at codon 717 (London mutations), also cause early-onset Alzheimer’s (26) but do so by a mechanism very different from that of the Swedish mutation.

After β-secretase cleavage, the C terminus of the β-peptide has to be generated by a further proteolytic event, which takes place in the TM domain of APP. In keeping with the imaginative and sequential nomenclature for the enzymes postulated to be involved in cellular APP proteolysis and Aβ generation, the enzyme cleaving in the TM domain to generate the C terminus of the Aβ peptide has been named γ-secretase.

It has been shown that most of the Aβ released from both cell lines derived from tissues other than those from the central nervous system and from neuronal cells terminates at residue 40. However, a small proportion (5–10%) extends to residue 42 (27). It has been postulated that the major pathologic culprit in Alzheimer’s disease is this subpopulation of Aβ, because this longer, more aggregation-prone species deposits preferentially in both sporadic and familial Alzheimer’s disease brains. Careful measurement of the Aβ released from cells transfected with the various London mutations revealed that although



The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement