I. INTRODUCTION: AN HISTORICAL PERSPECTIVE

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Few subjects in biomedicine have aroused the interest of the scientific and lay communities alike as has Alzheimer's disease (AD). The dramatic rise in life expectancy during the 20th century, from roughly 49 years to more than 76 years in the United States, has resulted in a burgeoning number of individuals achieving the age at which neurodegenerative disorders become common. Among these, AD has emerged as the most prevalent form of late-life mental failure in humans. It was not always so. When Alois Alzheimer, a Bavarian psychiatrist, first defined the clinicopathological syndrome that bears his name at a meeting in Munich in 1906, neither he nor his audience recognized that the disorder he described in a woman in her early 50s might ultimately turn out to be indistinguishable from common senile dementia. Indeed, it was not until the work of Blessed, Tomlinson, and Roth in the late 1960s that AD became generally accepted as the most common basis for senile dementia. We now recognize that a histopathological syndrome indistinguishable from that which Alzheimer originally described has an incidence which rises almost logarithmically with age. As a result, AD, originally believed to be a rare dementia occurring in the "presenile" period (that is, onset of symptoms under 65 years of age), is largely indistinguishable from senile dementia of the Alzheimer type, and the cases accrue at a linear rate rather than in a bimodal age distribution.

Alzheimer's original patient, a woman referred to as Auguste D. in his report, exemplified several cardinal features of the disorder that we still observe in most patients nowadays: progressive memory impairment; disordered cognitive function; altered behavior including paranoia, delusions, and loss of social appropriateness; and a progressive decline in language function. During the early and middle phases of this slow, inexorable process, the patient's alertness is well preserved, and motoric and sensory functions are essentially intact. However, as subjects continue to lose ground cognitively, slowing of motor functions such as gait and coordination often lead to a picture resembling extrapyramidal motor disorders such as parkinsonism.

For many decades after Alzheimer's original description, little progress in defining the pathogenesis of AD occurred. Although neuropathological studies led to growing recognition of the commonness of the syndrome, the study of AD and other idiopathic neurodegenerative disorders was marked by mechanistic ignorance and therapeutic nihilism. This situation began changing in the 1960s, when the advent of electron microscopy allowed Michael Kidd in England and Robert Terry in the United States to describe the striking ultrastructural changes underlying the two classical lesions which Alzheimer had linked: senile (neuritic) plaques and neurofibrillary tangles. In the mid 1970s, the first clear neurochemical clue as to what might underlie the dementing symptoms came from the observation that neurons synthesizing and releasing acetylcholine underwent variable but usually severe degeneration. This was observed as a decrease in the amounts and activities of the synthetic and degradative enzymes, choline acetyltransferase and acetylcholinesterase, in the limbic and cerebral cortices and an associated loss of cholinergic cell bodies in the subcortical nuclei that project to these regions, namely, the septal nuclei and the basal forebrain cholinergic system. As a result, substantial pharmacological research focused on attempting to enhance acetylcholine levels in the synaptic cleft, primarily by inhibiting the degradative enzyme. These efforts ultimately led to the only two drugs specifically approved to date for treating Alzheimer's disease in the United States: tetrahydroaminoacridine and donezepil.

In the late 1970s and early 1980s, variable deficits of other neurotransmitter systems were identified in AD brain tissue. It became increasingly clear that AD, unlike Parkinson's disease, did not involve degeneration of a single transmitter class of neurons but was highly heterogeneous. This realization appeared to explain the lack of robust clinical benefit in most patients treated with cholinergic drugs. Attention increasingly focused on attempting to identify the underlying mechanisms for the synpatic dysfunction and perikaryal degeneration that affected multiple classes of neurons in the limbic system and association cortices. In this context, investigators increasingly trained their sights on the two classical neuropathological lesions to which Alzheimer had called attention.

As neurochemists began attempting to identify the composition and molecular origin of the amyloid plaques and neurofibrillary tangles, they were reminded by their neuropathological colleagues that these lesions observed in the postmortem brain could be considered tombstones of the process that occurred late in disease and were thus unlikely to provide major insights into etiology and early pathogenesis. Thus one observed not only an ongoing debate about which of the two lesions might precede the other but a general sense that both lesions were likely to be preceded by some or many biochemical steps that might not become apparent simply by identifying the principal proteins composing these lesions. However, increasingly rapid scientific progress since the mid 1980s has proven these concerns ill-founded.

Advances in biochemical pathology, that is, the use of compositional analyses and immunocytochemistry to define the subunit composition of the plaques and tangles, were followed by signal advances in the molecular genetics of AD that have validated the critical role of the subunit proteins in the fundamental mechanisms of AD as well as certain other degenerative dementias. The elucidation of the genotype-to-phenotype relationships for each genetic alteration linked to familial forms of AD (a process which is still very active) has led to a growing consensus about how at least the familial forms of the disorder may begin. The result of this continuing work is that a rough temporal outline of the disease cascade has begun to emerge. This process and the closely related effort to identify points for therapeutic intervention have been markedly assisted by the development of imperfect but nonetheless highly useful cellular and animal models of the presumed early features of the disease mechanism.

In this article, I review the extensive neuropathological, biochemical, genetic, cell biological, and transgenic modeling studies that have contributed to our growing understanding of the etiopathogenesis of AD. Given the enormous amount of scientific activity directed toward this problem and to related basic biological questions, the review will perforce be selective, and the reader will be referred to primary literature and review articles that cover particular features of this complex topic. At the end, we consider the imminent initiation of therapeutic trials directed at certain key features of the disease cascade. If these "rational" treatment approaches engender some success, AD may emerge as a triumph of reductionist biology applied to a disorder of the most complex of physiological systems, the human cerebral cortex.

	II. DECIPHERING THE NEUROPATHOLOGICAL PHENOTYPE OF ALZHEIMER'S DISEASE

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Neuritic plaques, one of the two diagnostic brain lesions observed in Alzheimer's original patient, are microscopic foci of extracellular amyloid deposition and associated axonal and dendritic injury, generally found in large numbers in the limbic and association cortices (24). Such plaques contain extracellular deposits of amyloid -protein (A) that occur principally in a filamentous form, i.e., as star-shaped masses of amyloid fibrils. Dystrophic neurites occur both within this amyloid deposit and immediately surrounding it. These neurites are often dilated and tortuous and are marked by ultrastructural abnormalities that include enlarged lysosomes, numerous mitochondria, and paired helical filaments, the latter generally indistinguishable from those that comprise the neurofibrillary tangles (see below). Such plaques are also intimately associated with microglia expressing surface antigens associated with activation, such as CD45 and HLA-DR, and they are surrounded by reactive astrocytes displaying abundant glial filaments. The microglia are usually within and adjacent to the central amyloid core of the neuritic plaque, whereas the astrocytes often ring the outside of the plaque, with some of their processes extending centripetally toward the amyloid core. The time that it takes to develop such a neuritic plaque is unknown, but these lesions probably evolve very gradually over a substantial period of time, perhaps many months or years. The surrounding neurites that contribute to any one plaque can emanate from local neurons of diverse neurotransmitter classes. Much of the fibrillar A found in the neuritic plaques is the species ending at amino acid 42 (A₄₂), the slightly longer, more hydrophobic form that is particularly prone to aggregation (70). However, the A species ending at amino acid 40 (A₄₀), which is normally more abundantly produced by cells than A₄₂ (see below), is usually colocalized with A₄₂ in the plaque. The cross-sectional diameter of neuritic plaques in microscopic brain sections varies widely from 10 to >120 µm, and the density and degree of compaction of the amyloid fibrils which comprise the extracellular core also shows great variation among plaques.

When the A peptide that Glenner originally identified in meningovascular amyloid deposits from Alzheimer brains (35) was recognized as the subunit of the plaque amyloid (38, 96, 144), many laboratories developed sensitive antibodies to endogenous or synthetic A. Immunohistochemical staining with such antibodies revealed a far more extensive number of A deposits than had been appreciated by the use of classical silver impregnation methods, such as the Bielschowsky and Bodian stains. In retrospect, it became apparent that the most sensitive silver staining methods (e.g., the modified Bielschowsky stain and the Gallya's silver stain) could also recognize many A deposits that lacked the compacted, fibrillar appearance of the classical neuritic plaques. Many of the plaques found in limbic and association cortices, and virtually all of those in brain regions not clearly implicated in the typical symptomatology of AD (e.g., thalamus, caudate, putamen, cerebellum), showed relatively light, amorphous A immunoreactivity that occurred in a finely granular pattern, without a clearly fibrillar, compacted center. Moreover, staining with silver stains highly capable of recognizing dystrophic neurites (e.g., the Bodian method) as well as immunohistochemistry for various neuronal/neuritic cytoskeletal proteins indicated that there was very little or no detectable neuritic dystrophy in most of these amorphous-appearing, nonfibrillar plaques.

The recognition of these amorphous plaques in the late 1980s (71, 166, 199) and their detection in regions that also contained many neuritic plaques (i.e., limbic and association cortices) led to the concept that they might represent precursor lesions of neuritic plaques. These lesions were thus referred to as "diffuse" plaques or "preamyloid deposits." When it later was determined that the A peptides deposited in Alzheimer brain principally ended at either A₄₀ or A₄₂, it became apparent that peptides ending at A₄₂ were the subunits of the material comprising the diffuse plaques, with little or no A₄₀ immunoreactivity, in contrast to the mixed (A42 plus A40) deposits that generally were found in the fibril-rich neuritic plaques (68, 69, 85, 128a). The hypothesis that diffuse plaques represent immature lesions that are precursors to the plaques with surrounding cytopathology arose from two lines of evidence. First, diffuse plaques were the sole form found in those brain regions that largely or entirely lacked neuritic dystrophy, glial changes, and neurofibrillary tangles and were not clearly implicated in the typical clinical symptoms of AD, e.g., cerebellum, striatum, and thalamus. Second, healthy aged humans free of AD or other dementing processes often showed solely diffuse plaques in limbic and association cortices, i.e., in the same regions as Alzheimer patients showed mixtures of diffuse and neuritic plaques. The notion that diffuse plaques could be earlier lesions was later supported by studies of transgenic mice expressing mutant human APP. These mice usually showed diffuse deposits before developing fibrillar, thioflavin S-positive, and Congo red-positive neuritic/glial plaques. Indeed, this hypothesis was particularly well supported by immunohistochemical studies of patients with Down's syndrome (85). Such individuals often display diffuse deposits as early as their teenage years but do not show neuritic/glial plaques until some two decades later, a time at which they first display abundant neurofibrillary tangles in limbic and association cortices.

Many neurons in the brain regions typically affected in AD (entorrhinal cortex, hippocampus, parahippocampal gyrus, amygdala, frontal, temporal, parietal and occipital association cortices, and certain subcortical nuclei projecting to these regions) contain large, nonmembrane-bound bundles of abnormal fibers that occupy much of the perinuclear cytoplasm. Electron microscopy reveals that most of these fibers consist of pairs of ~10-nm filaments wound into helices (paired helical filaments or PHF), with a helical period of ~160 nm. Some tangle-bearing neurons also contain skeins of straight, 10- to 15-nm filaments interspersed with the PHF. Beginning in 1985, immunocytochemical and biochemical analyses of neurofibrillary tangles suggested that they were composed of the microtubule-associated protein tau (12, 43, 79, 107, 191). This was later confirmed by isolation of a subset of PHF that could be partially solubilized in strong solvents such as SDS (84) or digested with harsh proteases (75, 185), releasing tau proteins which migrated electrophoretically at a higher molecular weight than did normal tau prepared from tangle-free human or animal brains. This slower migration was shown to result from increased phosphorylation of tau; in vitro dephosphorylation with alkaline phosphatase returned this PHF-derived tau to essentially normal migration. Although some PHF can be solubilized by boiling in SDS (84), much of the tau in tangles is present in highly insoluble filaments (PHF) that are resistant to detergents such as SDS and chaotrophic solvents such as guanidine hydrochloride (145). Extensive analysis of the nature of hyperphosphorylated tau using antibodies specific for various phosphotau epitopes has helped clarify which residues are phosphorylated in PHF tau (37, 80, 84, 97) A variety of kinases have been shown to be capable of phosphorylating tau in vitro at various sites (e.g., Refs. 37, 65). Nevertheless, it has not become clear whether one or more kinases are principally responsible for initiating the hyperphosphorylation of tau in vivo that leads to its apparent dissociation from microtubules and aggregation into insoluble paired helical filaments. In this regard, a recent study provides evidence that a dysregulation of cyclin-dependent kinase 5 (cdk5), as a result of proteolytic cleavage of its regulatory subunit p35 to yield a fragment (p25) which allows constitutive activity of the kinase, could play a major role in the hyperphosphorylation of tau that appears to underlie tangle formation in AD (111). It has been reported that calpain is responsible for cleavage of p35 and that treating cells with A aggregates can trigger p35 activation and the subsequent cdk5-mediated phosphorylation of tau and perhaps other cytoplasmic substrates (83a).

The two classical lesions of AD, neuritic plaques and neurofibrillary tangles, can occur independently of each other. Tangles composed of tau aggregates that are biochemically similar to or, in some cases, indistinguishable from those in AD have been described in more than a dozen less common neurodegenerative diseases, in almost all of which one finds no A deposits and neuritic plaques. Conversely, A deposits can be seen in the brains of cognitively normal-aged humans in the virtual absence of tangles. There are also infrequent cases of AD itself which are "tangle poor," i.e., only a few neurofibrillary tangles are found in the neocortex despite abundant A plaques (168). It appears that in quite a few such cases, an alternate form of neuronal cytoplasmic inclusion, the Lewy body (composed principally of -synuclein protein), is found in cortical pyramidal neurons. In other words, the Lewy body variant of AD (not to be confused with diffuse Lewy body disease, which largely lacks A plaques) may represent a tangle-poor form of AD that still has the usual amount of A plaque formation (51). The fact that neurofibrillary tangles composed of altered, aggregated tau proteins occur in disorders (e.g., subacute sclerosing panencephallitis, Kuf's disease, progressive supranuclear palsy, etc.) in the absence of A deposition suggests that tangles can arise secondarily during the course of a variety of etiologically distinct neuronal insults. As we shall discuss, there is growing evidence that the formation of tangles in AD represents one of several cytological responses by neurons to the gradual accumulation of A and A-associated molecules.

Many of the dilated and tortuous neurites found within and immediately surrounding amyloid plaques contain PHF that are structurally, biochemically, and immunocytochemically indistinguishable from those that comprise the neurofibrillary tangles. In addition, plaques often contain numerous dystrophic neurites that are not immunoreactive for PHF tau. Tau-positive dystrophic neurites are also present in a more widespread distribution in the cortical neuropil outside of the neuritic plaques. The prevalence and density of dystrophic cortical neurites that contain altered forms of tau varies substantially among Alzheimer cases. There is evidence that cases that are particularly rich in neurofibrillary tangles are also those that show widespread tau-immunoreactive dystrophic cortical neurites (120). Some of the intraplaque and extraplaque dystrophic neurites are immunoreactive for phosphorylated forms of the neurofilament subunit proteins, and the latter can thus coexist with phosphotau reactivity. This finding suggests that there may be several substrates for the altered kinase and phosphatase (176) activities that occur in tangle-bearing neurons and dystrophic neurites.

A was originally isolated from amyloid-laden meningeal arterioles and venules that are often found just outside of the brains of patients with AD or Down's syndrome (34, 35). Similarly, small arterioles, venules, and capillaries within cerebral cortex also frequently bear amyloid deposits. This microvascular angiopathy is characterized at the ultrastructural level by amyloid fibrils found in the abluminal basement membrane of the vessels, sometimes with apparent extension or "spillover" of the fibrils into the surrounding perivascular neuropil (a lesion referred to as dyshorric angiopathy) (178). The A peptides that occur as filaments in the microvessel basement membranes appear, on the basis of immunoreactivity, to be principally A₄₀ species, although evidence has been presented that the initially deposited species in vessels destined to develop amyloid angiopathy may be A₄₂ (165). It is intriguing that meningeal arterioles that penetrate and traverse the cerebral cortex can have amyloid deposits in their walls that abruptly stop as the vessel enters the subcortical white matter. Only rare microvessels within the white matter show A deposits. The extent of amyloid angiopathy varies widely among AD brains that have relatively similar burdens of parenchymal (i.e., plaque associated) A. As a result, the contribution of this microvascular amyloidosis to the cortical dysfunction that occurs in AD and the mechanism by which amyloid alters microvascular function remain matters of active study (see for example Refs. 110, 172). Amyloid-bearing vessels composed of A deposits essentially indistinguishable from those of AD can occur in the virtual absence of parenchymal A deposits in the brains of elderly subjects without AD (178). Such amyloid-bearing vessels in this condition [referred to as congophilic amyloid angiopathy (CAA)], as well as those in AD can occasionally rupture, apparently due to hyaline necrosis surrounding the amyloid deposit in the vessel wall, leading to one or multiple cerebral hemorrhages (178). Nevertheless, the large majority of AD subjects do not experience cerebral hemorrhages, despite the presence of some or many microvascular amyloid deposits.

	III. ORIGIN OF AMYLOID -PROTEIN: CELL BIOLOGY OF -AMYLOID PRECURSOR PROTEIN

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The purification and partial sequencing of the A protein from meningovascular amyloid deposits in AD and Down's syndrome (34, 35) and the subsequent observation that A was also the subunit of the plaque amyloid (38, 96, 144) led to the cloning of the gene encoding the -APP (72). A is derived from its large precursor protein by sequential proteolytic cleavages (see sect. IIIB). APP comprises a heterogeneous group of ubiquitously expressed polypeptides migrating between 110 and 140 kDa on electrophoretic gels (146). This heterogeneity arises both from alternative splicing (yielding 3 major isoforms of 695, 751, and 770 residues) as well as by a variety of posttranslational modifications, including the addition of N- and O-linked sugars, sulfation, and phosphorylation (62, 108, 181, 183). The APP splice forms containing 751 or 770 amino acids are widely expressed in nonneuronal cells throughout the body and also occur in neurons. However, neurons express even higher levels of the 695-residue isoform, which occurs at very low abundance in nonneuronal cells (45). The difference between the 751/770- and 695-residue forms is the presence in the former of an exon that codes for a 56-amino acid motif that is homologous to the Kunitz-type of serine protease inhibitors (KPI), indicating one potential function of these longer APP isoforms. Indeed, the KPI-containing forms of APP found in human platelets serve as inhibitors of factor XIa, which is a serine protease in the coagulation cascade (158). APP is highly conserved in evolution and expressed in all mammals in which it has been sought. A partial homolog of APP is found in Drosophila (referred to as APPL) (130). Indeed, APP is a member of a larger gene family, the amyloid precursor-like proteins (APLPs) (157, 182), which have substantial homology, both within the large ectodomain and particularly within the cytoplasmic tail, but are largely divergent in the A region.

APP is a single transmembrane polypeptide that is cotranslationally translocated into the endoplasmic reticulum via its signal peptide and then posttranslationally modified ("matured") through the secretory pathway. Its acquisition of N- and O-linked sugars occurs rapidly after biosynthesis, and its half-life is relatively brief (~45-60 min in most cell types tested) (183). Both during and after the trafficking of APP through the secretory pathway, it can undergo a variety of proteolytic cleavages to release secreted derivatives into vesicle lumens and the extracellular space (Fig. 1). The first proteolytic cleavage identified, that made by an activity designated -secretase, occurs 12 amino acids NH₂-terminal to the single transmembrane domain of APP (28, 156). This processing results in the release of the large soluble ectodomain fragment (-APP_s) into the lumen/extracellular space and retention of an 83-residue COOH-terminal fragment (CTF) in the membrane. Alternatively, some APP molecules not subjected to -secretase cleavage can be cleaved by an activity designated -secretase, which principally cuts 16 residues NH₂-terminal to the -cleavage site, generating a slightly smaller ectodomain derivative (-APP_s) (147) and retaining a 99-residue CTF (C99) in the membrane that begins at residue 1 of the A region (reviewed in Ref. 143).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Schematic diagrams of the -amyloid precursor protein (APP) and its principal metabolic derivatives. Top diagram depicts the largest of the known APP alternate splice forms, comprising 770 amino acids. Regions of interest are indicated at their correct relative positions. A 17-residue signal peptide occurs at the NH2 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). A single membrane-spanning domain (TM) at amino acids 700-723 is indicated by the vertical dotted lines. The amyloid ß-protein (A) fragment includes 28 residues just outside the membrane plus the first 12-14 residues of the transmembrane domain. In the middle diagram, the arrow indicates the site (after residue 687; same site as the white dot in the A region of APP in the upper diagram) of a constitutive proteolytic cleavage made by protease(s) designated -secretase that enables secretion of the large, soluble ectodomain of APP (APPs-) into the medium and retention of the 83-residue COOH-terminal fragment in the membrane. The C83 fragment can undergo cleavage by a protease(s) called -secretase at residue 711 or residue 713 to release the p3 peptides. The bottom diagram depicts the alternative proteolytic cleavage after residue 671 by a protease(s) called -secretase that results in the secretion of the slightly truncated APPs- molecule and the retention of a 99-residue COOH-terminal fragment. The C99 fragment can also undergo cleavage by -secretase to release the A peptides.

Until 1992, it was assumed that A generation was a pathological event, because the cleavage of the C99 fragment resulting from the so-called -secretase activity appeared to occur in the middle of the transmembrane domain. It was assumed that this would require the release of C99 from the membrane, for example, as a result of some preexisting membrane injury that allowed access to a soluble protease. However, the use of sensitive A antibodies to probe the conditioned media of APP-expressing cells revealed secreted A that was constitutively released from cells under entirely normal cellular conditions (13, 50, 148, 151). This result suggested that the -secretase cleavage could be followed by a constitutive cleavage at the COOH terminus of the A region, made by an activity dubbed -secretase. At the same time, a peptide fragment designated p3 was discovered to be produced by the sequential actions of the - and -secretases (44, 50). These unexpected findings indicated that A production was a normal metabolic event, and indeed, the peptide was detected in both cerebrospinal fluid and plasma in healthy subjects throughout life (148, 151). Precisely where during its complex intracellular trafficking APP can undergo the -, -, and -secretase cleavages is not settled. Clearly, a substantial portion of -APP_s is generated by -secretase acting on plasma membrane inserted APP (155). On the other hand, -APP_s can also be generated during the secretory intracellular trafficking of APP (23, 133). With regard to the -secretase cleavage, this can occur in part late in the secretory trafficking of APP (49). The recent identification and cloning of -secretase by several laboratories (63, 153, 177, 201) will now enable a precise localization of this novel membrane-anchored aspartyl protease. The sites of cleavage of the C99 and C83 fragments by -secretase and the nature of that enzyme are also under active study. It appears that A₄₀ and A₄₂ can be made in considerable part during the internalization and endosomal processing of APP (77, 112). There are conflicting data about whether much of A₄₂ is generated early in the secretory trafficking of APP (i.e., in endoplasmic reticulum, intermediate compartment, and early Golgi) or principally after APP reaches the cell surface. Some evidence suggests that A peptides generated very early in the secretory pathway (i.e., in endoplasmic reticulum) may not be destined for secretion and are retained and catabolized inside cells (16). However, it is likely that the majority of A generated within cells is destined for secretion. Steady-state levels of A in human cerebrospinal fluid are in the range of 3-8 nM (101), whereas the level in plasma is generally under 500 pM (137). A₄₀ and A₄₂ species can both be detected in these extracellular fluids.

Pulse-chase experiments have demonstrated that most C83 and C99 fragments (the immediate substrates of -secretase) are generated from APP molecules that have undergone full N- plus O-linked glycosylation (i.e., within or after the Golgi) (49, 183). These results support the concept that the -, -, and -secretase cleavages of APP occur primarily at or near the cell surface, perhaps in substantial part in recycling endosomes (112).

In polarized epithelial cells such as Madin-Darby canine kidney (MDCK) cells, APP is principally targeted to the basolateral membrane, where it can undergo -secretase cleavage to release -APP_s basolaterally, although a small fraction is targeted and processed apically (47, 48). In neurons, which are one of the cells that express the highest levels of APP in the body (particularly APP₆₉₅), APP can be anterogradely transported in the fast component of axonal transport (76). APP is present in vesicles in axonal terminals, although not specifically in synaptic vesicles. Cell biological studies demonstrate that APP in the axonal terminals can be retrogradely transported up the axon to the cell body, and some molecules are then fully translocated to the somatodendritic surface (200). During its retrograde axonal trafficking, some APP molecules can apparently recycle to the axolemmal surface (200). Although it has been assumed that APP axonal terminals might be a principal site for the generation of A, this has not been definitively determined, and APP that recycles in endosomes at various neuronal subsites may be capable of undergoing the sequential - and -secretase cleavages to release the peptide. Indeed, although APP is particularly abundantly expressed in neurons and they have been directly shown to secrete substantial amounts of A peptides (50), other brain cells also express APP and release variable amounts of A, including astrocytes, microglia, and endothelial and smooth muscle cells, and these could all contribute to the secreted pool of A that eventually leads to extracellular deposition. Moreover, the fact that virtually all peripheral cells also express APP and generate A and that A is present in plasma raises the possibility that circulating A could cross the blood-brain barrier and contribute to cerebral A accumulation. Direct evidence that A can cross the blood-brain barrier in small amounts using a mechanism consistent with receptor-mediated endocytosis has been reported (116, 117, 209).

A number of possible functions have been ascribed to APP holoproteins and/or their major secreted derivative (-APP_s) based on cell culture studies. Soluble -APPs appear to be capable of acting as an autocrine factor (132) and a neuroprotective and perhaps neuritotrophic factor (98). The fact that the alternatively spliced forms containing 751 and 770 residues contain a 56-residue insert in the middle of the ectodomain encoding a KPI motif (167) has led to in vitro studies that confirm an ability of these isoforms to inhibit serine proteases such as trypsin and chymotrypsin (154). As mentioned previously, the KPI-containing isoforms also function as an inhibitor of factor XIa (a serine protease) in the clotting cascade (158). The secreted APP isoforms can confer cell-cell and cell-substrate adhesive properties in culture (e.g., Ref. 140). The APP holoprotein has also been suggested to function in cell-cell interactions when inserted at the plasma membrane, based on in vitro studies (122). All of these imputed functions have not yet been clearly confirmed in vivo. Deletion of the APP gene in mice results in neither early mortality nor appreciable morbidity; cerebral gliosis and changes in locomotor behavior occur later in adult life (207), and neurons cultured at birth have diminished viability and retarded neurite outgrowth (113). This lack of a vital consequence of APP deletion in vivo may result from the fact that mammals express proteins closely homologous to APP, the APLPs (157, 182). Delineation of the precise functions of APP and its homologs in vivo awaits further study. No evidence has emerged that a fundamental cellular function of APP is lost in AD patients. Instead, APP mutations seem to act by a toxic gain-of-function mechanism, namely, by increasing production of the potentially cytotoxic A fragment (see below).

	IV. GENETICS OF FAMILIAL ALZHEIMER'S DISEASE

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The first specific genetic cause of AD to be identified was the occurrence of missense mutations in APP (36) (Table 1). Despite extensive genetic surveying, such mutations have only been confirmed in some two dozen or so families worldwide. Nevertheless, the location of the mutations (Fig. 2) and the subsequent delineation of their genotype-to-phenotype relationships have provided critical insights into the mechanism of AD. The mutations are strategically located either immediately before the -secretase cleavage site, shortly after the -secretase site, or shortly COOH-terminal to the -secretase cleavage site. The fact that, despite substantial investigation, no other mutations in the large APP protein that cause AD have been discovered strongly suggests that these missense mutations lead to AD by altering proteolytic processing at the three secretase sites in subtly different ways. This hypothesis has been confirmed by analysis of each of the mutations, initially in transfected cells or primary cells from patients and then in transgenic mouse models (reviewed in Ref. 142). Families harboring APP missense mutations that cause AD generally have the onset of the disorder before age 65, often in their 50s.

View this table: [in this window] [in a new window]   Table 1. Confirmed genetic factors predisposing to Alzheimer's disease: relationships to the -amyloid phenotype

View larger version (98K): [in this window] [in a new window]   Fig. 2. -APP mutations genetically linked to familial Alzheimer's disease or related disorders. The sequence within APP that contains the A and transmembrane region is expanded and shown by the single-letter amino acid code. The underlined residues represent the A1-42 peptide. The vertical broken lines indicate the location of the transmembrane domain. The bold letters below the line indicate the currently known missense mutations identified in certain patients with familial Alzheimer's disease and/or hereditary cerebral hemorrhage with amyloidosis. Three-digit numbers refer to the residue number according to the -APP770 isoform.

There is another way that alterations in the APP gene can predispose to the development of AD. The overexpression of structurally normal APP owing to elevated gene dosage in trisomy 21 (Down's syndrome) almost invariably leads to the premature occurrence of classical AD neuropathology (neuritic plaques and neurofibrillary tangles) during middle adult years. A life-long increase in APP expression due to duplication of all of chromosome 21 or, in the case of translocation Down's syndrome, that portion of 21q containing the APP gene results in overproduction of A₄₀ and A₄₂ peptides dating from birth (173). This is assumed to be responsible for the strikingly early appearance of many A₄₂ diffuse plaques, which can occur as soon as age 12 yr (85). Down's subjects often display diffuse plaques composed solely of A₄₂ in their teens and 20s, with accrual of A₄₀ peptides onto these plaques and the appearance of associated microgliosis, astrocytosis, and surrounding neuritic dystrophy usually beginning in their late 20s or 30s (85, 94). This observation underscores the importance of A₄₂ accumulation as a seminal event in the development of AD-type brain pathology. The appearance of neurofibrillary tangles is also delayed until the late 20s, 30s, or beyond in most Down's patients. The gradual accrual of AD-type brain lesions in these individuals, who are retarded from birth for other reasons, appears to be associated in many cases with progressive loss of cognitive and behavioral functions after the age of 35 or so.

Because the entire chromosome 21 is duplicated in the vast majority of cases of Down's syndrome, it is difficult to attribute the Alzheimer syndrome that they develop directly to APP gene dosage. However, this issue has been essentially resolved by the recent evaluation of a patient with translocation Down's syndrome in which the obligate Down's region in the distal portion of chromosome 21 was duplicated, but the break point was telomeric to the APP gene. The subject bearing this particular translocation had typical phenotypic features of Down's syndrome but did not develop clear-cut evidence of behavioral deterioration during middle age. At autopsy, no significant A deposition or other Alzheimer-type neuropathology was observed (119). This absence of amyloid deposition and attendant cytopathological changes is highly unusual in Down's subjects, and this case suggests that when this occurs, it is because the APP gene is not duplicated. The careful clinicopathological analysis of this unusual case provides further strong support for the primacy of A deposition in producing classical AD neuropathology.

The realization that autosomal dominant AD is genetically heterogeneous led to intensive searches for loci in the genome besides APP that could explain the many families that did not link to chromosome 21. Establishment of a linkage of some of these families to chromosome 14 (135) led ultimately to further linkage analysis and positional cloning that identified a novel gene on chromosome 14q which came to be known as presenilin 1 (PS1) (150). Missense mutations were found that appeared to be causative of AD in certain families with clinical onset in their 40s and 50s, sometimes as early as the 30s. Shortly thereafter, an homologous gene was discovered on chromosome 1, mutations in which explain the early-onset kindreds referred to as the Volga German families, as well as AD in an Italian family (90). This gene was ultimately designated presenilin 2 (PS2). Further intensive genetic surveys have identified as many as 75 missense mutations in presenilin 1 and three in presenilin 2 as molecular causes of early-onset AD in several hundred families worldwide (reviewed in Ref. 52). Presenilin 1 missense mutations cause the earliest and most aggressive form of AD, commonly leading to onset of symptoms before the age of 50 and demise of the patient in his/her 60s. We discuss below how instructive these mutations have been for understanding both the role of presenilin in AD and gaining insight into the normal functions of these interesting polytopic membrane proteins.

Whereas the autosomal dominant mutations in APP or the presenilins are quite infrequent causes of AD, the discovery that the 4 allele of apolipoprotein E (ApoE) predisposes to AD provided a major genetic risk factor for the disorder in the typical late-onset period (163). Studies initiated by searching for proteins in human cerebrospinal fluid that could bind immobilized A peptides on a filter led to the identification of ApoE as such a protein and the recognition that its gene localized to chromosome 19q, in a region previously found to show genetic linkage to AD in some late-onset families (163). Further genetic analyses indicated that the 4 allele of ApoE is overrepresented in subjects with AD compared with the general population and that inheritance of one or two 4 alleles heightens the likelihood of developing AD and makes its mean age of onset earlier than in subjects harboring 2 and or 3 alleles (18, 134). Thus the ApoE4 protein helps precipitate the disorder primarily in subjects in their 60s and 70s. There is also evidence that inheritance of the 2 allele may confer protection against the development of AD (17). Although inheritance of a single 4 allele may increase the likelihood of developing AD in the 60s and 70s, some two- to fivefold and two 4 alleles may increase the risk well above fivefold, it should be emphasized that ApoE4 is a risk factor for, not an invariant cause of, AD. Some humans homozygous for the 4 isoform still show no Alzheimer symptoms in their ninth decade of life and beyond. Conversely, a great many humans develop AD without harboring 4 alleles. The recognition that inheritance of 4 predisposes humans to AD provided one of the first genetic risk factors for a common late-onset disease.

Whereas there is universal agreement that alterations in the four aforementioned genes can cause familial forms of AD, various methods of genetic analysis indicate that additional genes predisposing to AD exist. In this regard, an AD-linked locus on chromosome 12 in certain pedigrees appears to represent alterations in or near the gene encoding ₂-macroglobulin (₂M) (10). A polymorphism in an intronic region of the ₂M gene segregates with the AD phenotype in some late-onset subjects (10). Additional studies confirming this association have appeared (2, 25, 102, 129), and work to determine whether the enhanced genetic risk is attributable directly to ₂M or to a nearby gene is underway.

The fact that numerous families exist whose AD phenotype does not link to any of the five genes implicated to date indicates that additional genetic risk factors and perhaps even dominantly transmitted causative genes will be found. Indeed, recent studies have revealed an apparent major locus for late-onset familial AD on chromosome 10q (8a). It is likely that over the next one to two decades, a much larger portion of AD will be shown to have genetic determinants than is currently believed. Indeed, clinical surveys already indicate that, upon careful questioning, a family history of first degree relatives with a dementing syndrome resembling AD is obtained in as many as one-half to two-thirds of patients presenting with clinically probable AD.

	V. GENOTYPE-TO-PHENOTYPE CONVERSIONS IN FAMILIAL ALZHEIMER'S DISEASE

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The rapid accrual of information about the proteolytic processing of APP and the aggregational properties of its A derivatives coincided with the identification of gene defects that cause or predispose to AD. The systematic correlation of these two distinct bodies of knowledge during the last few years has led to an emerging understanding of the fundamental pathogenetic mechanism of AD. Experiments to decipher the genotype-to-phenotype relationships have been conducted in cell culture, in transgenic mice and, most importantly, in patients who actually harbor the relevant genetic mutations. For all four genes unequivocally confirmed to date (APP, ApoE4, PS1, and PS2), inherited alterations in the gene products have been credibly linked to increases in the production and/or the cerebral deposition of the A peptides (142). Such studies have provided the strongest support for the hypothesis that cerebral accumulation of A is an early, invariant, and necessary event in the genesis of AD.

The nine known missense mutations in APP currently linked to familial AD (Fig. 2) have been found to increase A production by subtly different mechanisms. A double mutation in the two amino acids immediately preceding the -secretase cleavage site (often referred to as the "Swedish" APP mutation based on the ethnic origin of the family in which it occurs) induces increased cleavage by -secretase to generate more A₄₀ and A₄₂. The five mutations occurring just COOH-terminal to the -secretase cleavage sites appear in slightly different ways to selectively enhance the production of A species ending at residue 42. The two remaining mutations that are located internally in A could be expected to enhance the aggregational properties of all A species, although this has only been shown for the E693Q mutation that causes hereditary cerebral hemorrahge with amyloidosis of the Dutch type (89). The other, immediately adjacent internal mutation (A692G) leads to a mixed phenotype of 1) AD-type plaque and tangle formation associated with dementia and 2) severe microvascular -amyloidosis with occasional cerebral hemorrhages (56). This mutation has been shown in transfected cells to lead to changes in the heterogeneous NH₂-terminal -secretase cleavages that have the overall effect of favoring production of the full-length peptide beginning at A Asp1(46). The recent cloning of -secretase, a novel membrane-anchored aspartyl protease with its active site in its ectodomain and the identification of a close homolog thereof (BACE-2) (63, 153, 177, 201), has led to studies in transfected cells suggesting that the A692G APP mutation specifically enhances the proportion of APP that is cleaved by BACE-2 and shifts cleavage by the latter toward the Asp1 NH₂ terminus (M. Farzan, personal communication). These interesting early data suggest that the identification of the actual -, -, and -secretases will provide much clearer mechanistic insights into exactly how missense mutations in APP lead to heightened production of various A species, in each case inducing an amyloidogenic phenotype that produces AD.

Perhaps the most intriguing genotype-to-phenotype relationships in AD involve the presenilin mutations. When presenilin 1 and 2 were first cloned, the mechanism by which mutations in them produced the AD phenotype was an open matter and was not necessarily expected to involve enhanced A production. However, direct assays of A₄₀ and A₄₂ in the plasma and the cultured skin fibroblast media of humans harboring these mutations soon revealed a selective approximately twofold elevation of A₄₂ levels (137). Extensive modeling of these mutations in cultured cells and transgenic mice has confirmed this finding (e.g., Refs. 11, 15, 26, 174, 195). A particularly important observation has been the finding that crossing mice transgenic for human APP with mice expressing a PS1 missense mutation leads to a substantially accelerated AD-like phenotype in the offspring, with A₄₂ plaques (first diffuse and then mature) occurring as early as 3-4 mo of age (57). But even before confirmation of the A₄₂-elevating effect of presenilin mutations was obtained in transfected cells and transgenic mice, quantitative image analysis of the brain amyloid deposits of patients who had these mutations using A₄₂- and A₄₀-specific antibodies demonstrated directly that inheritance of presenilin mutations leads to a 1.5- to 3-fold increase in the relative abundance of plaques containing A₄₂ peptides, compared with the levels observed in sporadic cases of AD (86, 95). The molecular mechanism by which missense mutations in the presenilins selectively increase the -secretase cleavage of C99 (and also C83) to yield more peptides ending at A₄₂ compared with A₄₀ will be discussed after reviewing current knowledge about the complex biology of the presenilins (see sect. VI).

Even before ApoE4 was recognized as a genetic risk factor for late-onset disease, immunohistochemistry had demonstrated the presence of ApoE protein in a high percentage of A deposits in AD brain tissue (103). Once the genetic connection between AD and ApoE4 inheritance was made, further immunohistochemical studies of brains of patients lacking or expressing the ApoE4 protein showed that inheritance of ApoE4 was associated with a significantly higher A plaque burden than was observed in patients lacking ApoE4 (31, 126, 138). Although some brains of ApoE4 allele carriers showed higher neurofibrillary tangle densities, overall this change did not usually reach the statistically significant levels of elevation observed for A deposits. Importantly, studies in nonogenarians who died without showing clear-cut clinical symptoms of AD demonstrated that ApoE4 genotype was again linked to enhanced amounts of diffuse A₄₂ plaques in the brain, suggesting that the A-elevating effects associated with ApoE4 inheritance could be observed presymptomatically or in hosts who would not necessarily develop AD (118).

The mechanism by which ApoE4 protein leads to increased A deposition has been difficult to pinpoint. No evidence has emerged that A production is significantly elevated in cells that coexpress APP with the ApoE4 protein versus with the ApoE2 or ApoE3 proteins (9). Rather, ApoE4 seems to enhance the steady-state levels of A peptides, A₄₀ in particular (31), presumably by decreasing its clearance from the brain tissue in some way. In vitro studies quantifying the degree of A fibrillogenesis using synthetic peptides suggest that the presence of the ApoE4 protein results in increased numbers of fibrils, compared with levels obtained in the presence of ApoE3 (29, 93), although the way in which ApoE proteins cause these effects, e.g., by ApoE4 serving as a less effective inhibitor of A fibrillogenesis or rather as a more potent stimulator, is not settled. An alternative mechanism for the AD-promoting effect of ApoE4 inheritance emerges from evidence in transgenic mice expressing either the E4 or E3 human protein. Mice expressing E4 appear to have decreased neuritic outgrowth of cultured neurons and decreased maintenance of established neurites (105). These studies suggest that ApoE4 protein is less supportive of normal neuronal form and function than are ApoE3 or E2 proteins. However, such a neuronal vulnerability in ApoE4 gene carriers may not be the actual mechanism of the ApoE4 effect on the AD phenotype, given the fact that deposition of A into cerebral and meningeal vessels to produce the syndrome of congophilic amyloid angiopathy is also enhanced by the gene dosage of ApoE4, even in the absence of Alzheimer-type neuropathology (40). In other words, the fact that ApoE4 alleles have clearly been found to enhance A deposition not only in parenchymal plaque deposits but also in microvessels outside of the brain parenchyma (and in the absence of AD) potentially separates the ApoE4 effect in promoting the AD cerebral phenotype (i.e., neuritic plaques) from any deleterious effects ApoE4 may have on neuronal/neuritic function in general. Thus the most parsimonious explanation for ApoE4 effects vis á vis AD is that this isoform somehow enhances the deposition or decreases the clearance of A peptides, particularly A₄₀, in both the cerebral cortex and its microvasculature.

Such an amyloid-enhancing mechanism is supported by studies in which mice transgenic for mutant human APP are crossed with mice in which the endogenous mouse ApoE gene is deleted. The resultant offspring show substantially decreased A plaque burden compared with that seen in the parental APP transgenic line, suggesting that the absence of ApoE significantly decreases the tendency of A to accrue as diffuse and mature plaques (5). Moreover, mice lacking endogenous ApoE that express human ApoE3 or E4 plus mutant human APP develop less A deposits than similar mice expressing no ApoE at all (58).

An important caveat about in vitro studies attempting to elucidate the mechanisms by which the ApoE proteins induce such effects is that they need always to be conducted in the presence of lipid, i.e., where ApoE is assembled into lipoprotein particles. There is currently no evidence that any significant portion of ApoE proteins occurs as free polypeptides in brain or other tissues. As a result, early studies examining the effects of pure ApoE on A in vitro are difficult to interpret. Carefully designed in vitro and in vivo experiments should ultimately clarify whether ApoE4 increases A steady-state levels in brain by less efficiently preventing its aggregation, by inhibiting its degradation or its reuptake into cells, or by other effects on its clearance.

	VI. FUNCTION OF PRESENILINS: A CENTRAL ROLE IN INTRAMEMBRANOUS PROTEOLYSIS

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Shortly after the PS1 and PS2 genes were cloned and missense mutations within them shown to cause autosomal dominant AD, two important observations about their biology were made. First, the presenilin holoproteins (~44 kDa) were found to undergo constitutive endoproteolysis in many cell types and in the brain and thus exist in major part as stable heterodimers composed of the NH₂-terminal fragment (NTF) and COOH-terminal fragment (CTF) (11, 115, 123, 169). The very low levels of holoprotein in cells and tissue, together with the evidence from pulse-chase experiments that the holoprotein is rapidly converted into fragments (115), probably by endoproteolysis occurring within endoplasmic reticulum vesicles and subsequent stabilization of the fragments in the Golgi (206), suggests that the fragments are the principal biologically functional form of presenilins. The constitutive proteolytic cleavage site (115, 162) occurs within a hydrophobic portion of the cytoplasmic loop between the sixth and seventh of the eight putative transmembrane domains (91). The steady-state levels of presenilin NTFs and CTFs seem to be tightly regulated, as overexpression of PS1 in transfected cells or transgenic mice generally does not increase the overall level of PS fragments (11, 170). Excess PS holoproteins are rapidly degraded, mainly by the proteasome (73, 161). Once formed, PS fragments can associate into higher molecular mass (~100-200 kDa) complexes that may represent the principal form in which presenilin functions in cells (14, 205).

The second major observation was the identification of the homolog in Caenorhabditis elegans of the mammalian presenilins, a gene designated sel-12 (88). Sel-12 was identified in genetic screens as a facilitator of the worm homolog of Notch, lin-12. The existence of mutations in sel-12 that decrease or eliminate its function has enabled the use of the nematode as a model system for studying the function of the human presenilins (6, 87). For example, a loss-of-function mutation in sel-12 can produce a lethal defect in egg-laying in the worm that is due to a defect in lin-12 (i.e., Notch) signaling during differentiation of the vulva (88). Other proteins that interact genetically with sel-12 have been identified in C. elegans (192). In addition, the use of the yeast two-hybrid system has led to identification of several novel or known mammalian proteins that appear to interact with presenilin. Prominent among these are members of the Armadillo family called the catenins, including an apparent neuron-specific member of this family, designated -catenin (205, 208). Both - and -catenins coimmunoprecipitate with presenilin 1. The catenin binding site appears to be in the distal portion of the large cytoplasmic loop between transmembrane (TM) domains 6 and 7. It has been shown that this region is dispensable for the function of presenilin in the -secretase mechanism (i.e., in A generation), and therefore, the interaction with the catenins may not turn out to have pathogenic relevance in AD. Presenilins have recently been shown to participate in multi-protein complexes near and at the cell surface that include the cadherins, important molecules mediating cell-cell adhesion (33). Furthermore, the fact that mutations of conserved residues in PS1 as well as PS2 can elevate A₄₂ production and are linked to familial AD suggests that sequences that diverge between the two homologs (such as the region of the PS1 loop which binds the catenins) are less likely to be required for the critical stabilization of the presenilin heterodimers and for their AD-promoting activity than are highly conserved sequences, such as their COOH termini (175). Indeed, the latter site is a good candidate for the binding of the currently unknown cellular factors that regulate presenilin endoproteolysis and stabilize the heterodimers (198).

The loss of function of presenilin produced by gene deletion in mice leads to a profound phenotype that includes markedly abnormal somitogenesis and axial skeletal development with shortened body length, as well as cerebral hemorrhages (149, 190). In addition, these mice, which die just before or at birth, show abnormal embryonic neurodevelopment in the forebrain marked by premature loss of neuronal precursors (149). Deletion of just one PS1 gene in the mouse has not been associated with any major phenotypic abnormalities to date. An important functional insight has been gained by complementation studies in presenilin homozygous knockout mice. Crossing presenilin heterozygous knockout mice with mice transgenic for AD-causing mutant PS leads to some offspring that have no endogenous (mouse) presenilin but express the human mutant form. Such mice survive and do not have the devastating phenotype found in presenilin homozygous mice, although they may have subtle alterations (20, 121). Therefore, missense mutations in the human presenilins that cause early-onset AD appear to act as gain rather than loss of function mutations.

Presenilin knock-out mice have also proven to be critical for deciphering the role of presenilins in APP metabolism. Such mice show normal levels of APP holoproteins as well as normal secretory derivatives from the - and -secretase cleavages but grossly abnormal -secretase function (22). Neurons cultured from these mice (22) and the brain tissue itself (196) accumulate high levels of the -secretase APP substrates C83 and C99. There is a corresponding substantial (~70%) decrease in the production of both A₄₀ and A₄₂ (22). This evidence that presenilin plays a required role in the -secretase mechanism has received substantial support from several types of experiments. Even before the realization that presenilin is necessary for proper -secretase cleavage of APP, it was shown that presenilin could bind to and immunoprecipitate with full-length APP molecules in several cell types (184, 197). This interaction was shown not to require the cytoplasmic tail of APP (197). Because the presenilins have very small ectodomain loops, it was unlikely that presenilin and APP would interact via their respective ectodomains. This left the transmembrane domains as the likely site of interaction. However, this evidence for coimmunoprecipitation of presenilin and APP was sharply challenged by investigators who could show no such interaction (171). From this controversy arose two broad hypotheses for the mechanism of the presenilins in -secretase-mediated APP processing. The first, based on the ability to coprecipitate the proteins, suggested that presenilin participates directly in the -secretase mechanism, i.e., is part of the catalytic complex, presumably as a cofactor (197). The alternate hypothesis argued that presenilin and APP do not physically interact; rather, presenilin regulates the membrane trafficking of certain proteins, presumably including the components of the -secretase reaction (the protease and APP) in a way that allows them to come together (104, 171). In the author's laboratory, confirmation of the presenilin-APP interactions in multiple experiments and evidence that the two fractionate to the same enriched vesicular fractions on gradients (196, 206) and that the subcellular distribution of C83 and C99 (the immediate substrates of the -secretase reaction) was not altered in mice bearing or lacking PS1 suggested that presenilin was inseparable from the -secretase cleavage event, i.e., that it is a physical participant rather than having an indirect role via membrane trafficking.

In a separate line of work, Wolfe and colleagues (186, 187) designed peptidomimetic transition state ana