Biology of the Amyloid PProtein and Its Precursor Polypeptide

AP has provided a starting point for molecular biological and genetic studies that led to the eventual identification of the first specific molecular cause of AD—missense mutations in and around the AP region of the P-amyloid precursor protein (APP). APP, an intriguing and now much studied polypeptide

Fig. 1. Schematic diagrams of the P-amyloid precursor protein and its principal metabolic derivatives. (Top) The largest of the known APP alternate transcripts, comprising 770 amino acids. Regions of interest are indicated at their correct relative positions. A 17-residue signal peptide occurs at the amino terminus (box with vertical lines). Two alternatively spliced exons of 56 and 19 amino acids are inserted at residue 289; the first contains a serine protease inhibitor domain of the Kunitz type (KPI). Two sites of N-glycosylation (CHO) are found at residues 542 and 571. A single membrane-spanning domain at amino acids 700-723 is indicated by the vertical hatched bar. The amyloid p-protein (Ap) fragment (white box) includes 28 residues just outside the membrane plus the first 12-14 residues of the transmembrane domain. (Middle) The arrow indicates the site (after residue 687) of a constitutive proteolytic cleavage made by an unknown pro-tease(s) designated a-secretase that enables secretion of the large, soluble ectodomain of PAPP (APPs) into the medium and retention of the 83-residue carboxy-terminal fragment ~10-kDa) in the membrane. The 10-kDa fragment can undergo cleavage by an unknown protease(s) called 7-secretase at residue 711 or residue 713 to release the p3 peptides. (Bottom) The alternative proteolytic cleavage after residue 671 by an unknown en-zyme(s) called p-secretase that results in the secretion of a truncated APPs molecule and the retention of a 99-residue (~12-kDa) carboxy-terminal fragment. The 12-kDa fragment can also undergo cleavage by 7-secretase to release the Ap peptides.

(Fig. 1), is a large glycoprotein anchored in various cellular membranes (including the plasma membrane) by a single transmembrane region. It thus projects from the cell surface (and also into the lumens of many intracellular vesicles) in a fashion resembling well-characterized receptors such as the low density lipoprotein receptor and the insulin receptor. APP is widely expressed in virtually all mammalian cells. In the nervous system, neurons show particularly high expression, but astrocytes, microglia, and endothelial cells also express the precursor. The localization of the APP gene to chromosome 21q is widely believed to explain the observation that patients with trisomy 21 (Down's syndrome) incur p-amyloid deposition as early as late childhood and gradually develop the classical neuropathological features of AD by age 40 or so (16-18).

The primary structure of APP (10) shows us that the 40-42 residue Ap peptide that constitutes the amyloid actually comprises the 28 amino acids immediately outside of the single transmembrane region plus the first 12 or 14 amino acids of that membrane-buried segment (Fig. 1). This topography of the Ap region led to the assumption that an insult to cell membranes must occur before Ap could be released intact into the extracellular space of the brain to form amyloid deposits. In turn, this concept seemed consistent with the widely held opinion that tissue amyloid deposits in general were likely to represent secondary byproducts of disease processes rather than serving as an initiating feature which could be linked to the genetic etiology of a disease. However, extensive studies of systemic amyloid diseases as well as AD have shown that this concept is erroneous.

As investigators examined cultured cells that express APP naturally or were transfected with its cDNA to achieve high expression, they found that APP commonly undergoes a proteolytic cleavage just 12 amino acids in front of the membrane-anchoring region, that is, immediately after amino acid 16 of the Ap region of the precursor (19, 20) (Fig. 1). This scission releases the large, soluble ectodomain (referred to as APPs) into the extracellular fluid. The cleavage is caused by an as yet unidentified protease(s) that is referred to as "a-secretase." The APPs derivative has been found in normal human CSF (21,22) and plasma (23). Although a few studies have suggested that its level might be decreased in AD, most studies have found that this change is inconsistent enough as to not be diagnostically useful.

The APPs that is constitutively secreted by most cells in the body must serve one or several normal functions. Some of these have been suggested by studies in tissue culture. The normal functions of APPs may include: 1) the inhibition of certain serine proteases (e.g., trypsin, chymotrypsin and factor XIa of the coagulation cascade); 2) the participation in the adhesion of some cell types to the extracellular matrix; and 3) trophic, neuroprotective, and wound-healing properties. In addition, the uncleaved APP holoprotein residing at the cell surface may have its own function, for example, as a molecule that promotes cell-cell interactions or perhaps as a receptor for an as-yet-unknown diffusable ligand.

Although clues to the normal function of APP have emerged from cell culture studies, the use of genetic engineering to entirely delete ("knock out") the APP gene in mice has shown that the gene is not necessary for viability and normal brain development and that the phenotypic consequences are relatively subtle (24). Further study is needed before we can be certain of the functions of APP in the normal nervous system in vivo. Nevertheless, there is no compelling evidence that any putative function of APP is actually lost or diminished in AD subjects. Rather, it appears that the role of APP in AD involves a toxic function imparted by just its Aß fragment, once it is released from the precursor by proteolysis and begins to aggregate.

A major reinterpretation of our understanding of Aß came from the discovery in 1992 that APP can be alternatively metabolized in a way that avoids a-secretase cleavage within the Aß region and instead produces cleavages at the beginning of the Aß region [by a protease(s) dubbed "ß-secretase"] and at the end of this region [by a protease(s) designated "7-secretase"] (25-27) (Fig. 1). In other words, it was found that Aß is constitutively released from a subset of APP molecules during normal cellular metabolism, without any requirement for preexisting membrane injury or another form of cell damage. Indeed, it was found that intact 40- and 42-residue Aß peptides were normally present in extracellular fluids such as plasma and CSF (26,27). Moreover, APP-expressing cells cultured in the laboratory (neurons, astrocytes, fibro-blasts, and kidney cells, to name a few) all normally secreted Aß into the culture medium (25-28). These unanticipated findings brought the ß-amy-loidosis of AD in line with a number of known human amyloid deposition diseases outside of the brain, such as familial amyloidotic polyneuropathy (due to transthyretin amyloidosis) and secondary amyloid deposits (derived from the acute-phase protein, serum amyloid A), that arise in several inflammatory disorders. In virtually all of the amyloidotic diseases of humans, a circulating protein or protein fragment that is normally present in extracellular fluids undergoes progressive polymerization into amyloid fibrils, which form multiple tissue deposits capable of exerting local cytotoxicity (29).

There are at least three major implications of the discovery of normal Aß secretion for the study of AD (30). First, any genes that are implicated in the etiology of AD can be studied as to their effect on Aß production, both in transfected cells and transgenic mice bearing a mutant gene and in the CSF and plasma of patients carrying the mutation. Second, the levels of Aß40 and Aß42 can be directly assayed in plasma and CSF to determine whether they were altered in amount and thus are diagnostically useful in subjects with AD. Third, and perhaps most important, cell lines expressing normal or mutant APP and thus secreting Aß can serve as an in vitro screening system to identify compounds which specifically lower Aß production without damaging the cells. "Hits" in this assay can then be tested in animals (e.g., normal or transgenic mice) to determine whether they lower Aß production in vivo. As we will see, all of these principal implications of the discovery of soluble Aß production have now been realized.

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