1
MINIREVIEW:
Amyloid -Protein and the Genetics of Alzheimer's Disease*
Dennis J. Selkoe
From the Department of Neurology and Program in Neuroscience, Harvard Medical School and Center for
Neurologic Diseases, Brigham and Women's Hospital, Boston, Massachusetts 02115
This article was published in the Journal of Biological Chemistry, Volume 271, Number 31, Issue of
August 2, 1996 pp. 18295-18298
INTRODUCTION
The effort to decipher the mechanism of Alzheimer's disease (AD)1 has attracted the
interest of investigators from diverse biological disciplines, including biochemistry, cell
biology, molecular genetics, neuroscience, and structural biology. The eclectic nature of
research approaches to AD and the intensity of scientific interest in the problem have
made it increasingly likely that AD will become a premier example of the successful
application of biological chemistry to the identification of rational therapeutic targets in a
major human disease. Much of the recent progress in elucidating the pathogenesis of AD
has centered on the apparent role of the 40-42-residue amyloid -protein (A ) (1) as a
unifying pathological feature of the genetically diverse forms of this complex disorder.
Biochemistry of Cerebral -Amyloidosis, a Route to Genetic Insights
It has been known since the time of Alzheimer (2) that insidiously progressive loss of
memory, cognition, and behavioral stability in older humans can be associated with the
development of innumerable intraneuronal and extracellular filamentous lesions in the
limbic and cerebral cortices. Inside neurons, bundles of abnormal ~20-nm cytoplasmic
fibers (paired helical filaments) occur both in neuronal cell bodies (comprising
neurofibrillary tangles) and in axons and dendrites (referred to collectively as dystrophic
neurites). In addition to this filamentous degeneration of selected neurons and their
processes, AD is characterized by abundant extracellular masses of ~8-nm filaments
composed of A . These spherical deposits of A fibrils (amyloid plaques) are often
intimately associated with dystrophic axons and dendrites (some of which contain paired
helical filaments) as well as with activated microglia and reactive astrocytes. The
presence of such ``neuritic plaques,'' together with numerous neurofibrillary tangles, in
the hippocampus, amygdala, cerebral cortex, and certain other brain regions serves as the
basis for a definitive pathological diagnosis of AD. It should be noted that the amyloid protein of AD is only one of several different proteins that can accumulate excessively in
the extracellular spaces of various tissues and produce distinct human diseases
(collectively called amyloidoses).
Although arguments were once raised that studying the biochemistry of the plaques and
tangles would be unlikely to lead to insights into critical events in AD pathogenesis, this
has not turned out to be the case. Immunocytochemical and biochemical analyses of the
intraneuronal neurofibrillary tangles conducted during the last decade have led to the
2
conclusion that the microtubule-associated phosphoprotein, tau, is the major or, more
likely, the sole subunit of the paired helical filaments found in both the tangles and in
many of the dystrophic neurites observed in AD cortex (3, 4). Intensive studies by
numerous laboratories have shown that tau protein, which normally enhances the
polymerization of tubulin into microtubules and stabilizes these organelles in neurons,
becomes excessively phosphorylated, apparently due to a combination of enhanced
activity of certain kinases and decreased activity of certain phosphatases. Thus, the
modification of this normally soluble neuronal protein into an insoluble filamentous
polymer seems to involve a disregulation of cytoplasmic
phosphorylation/dephosphorylation cascades, but the factors that trigger this imbalance
are poorly understood. Importantly, neurofibrillary tangles composed of paired helical
filaments containing hyperphosphorylated tau molecules are found in a variety of
etiologically diverse neurological diseases besides AD, strongly suggesting that this
cytoskeletal alteration can develop as a secondary (albeit important) response to a variety
of cerebral insults. In accord with this view, the gene on human chromosome 17 that
encodes the tau polypeptides is not known to be the site of disease-causing mutations in
familial forms of AD.
In contrast, studies of A , which is the subunit of the amyloid fibrils found in neuritic
plaques and in some meningeal and cerebral blood vessels, resulted in the identification
of the first specific genetic cause of AD. The purification of both plaque and vascular
amyloid deposits and the isolation of their ~40-residue constituent peptide (A ) led to the
cloning of the type 1 integral membrane glycoprotein from which A is proteolytically
derived, namely the -amyloid precursor protein ( APP) (5). The localization of the APP
gene to chromosome 21q appeared to explain the observation that patients with trisomy
21 (Down's syndrome) incur -amyloid deposits in late childhood or young adulthood and
subsequently develop the classical neuropathological features of AD in their forties (6, 7,
8, 9). This realization led in turn to a specific search for families with autosomal
dominant AD who had genetic linkage to chromosome 21, resulting ultimately in the
identification of six different missense mutations in APP, five associated with familial
AD (10, 11, 12, 13, 14) and one with the neuropathologically related syndrome of
hereditary cerebral hemorrhage with amyloidosis of the Dutch type (15). Although APP
mutations, all of which are clustered in the A region of the precursor, have proven to be
a very rare cause of familial AD, representing less than 1% of such cases, they are
important for understanding the mechanism of the progressive cerebral A deposition that
occurs in all cases of AD.
Most of the APP missense mutations have now been examined in transfected human cell
lines, and their phenotypic effects have also been studied directly in primary skin
fibroblasts and plasma obtained from subjects harboring these mutations. In each
example studied to date, the missense mutations have been shown to alter the proteolytic
processing of the precursor (see below) in a way that results in increased production of
A peptides, particularly of the highly hydrophobic (and thus amyloidogenic) 42-residue
form of A (A 42) (16, 17, 18, 19, 20). For example, one of the disease-causing missense
mutations occurs immediately N-terminal to the start of the A region, i.e. at the P1
position for the protease (called `` -secretase''), which generates the N terminus of A
3
(Fig. 1). Other mutations occur 4 residues C-terminal to the end of the A region and lead
to increased cleavage of APP by the protease (called `` -secretase'') that generates the C
terminus of the A 42 peptide (Fig. 1). The elucidation of the effects of the APP mutations
on cellular A production was made possible by the discovery in 1992 that A is
proteolytically generated from APP under normal metabolic conditions and is
constitutively secreted by essentially all neural and non-neural cells that express the
precursor (21, 22, 23). The heightened cerebral deposition of A that results from the
effects of the APP missense mutations can be viewed as loosely analogous to the
excessive tissue deposition of another normal metabolite, cholesterol, in subjects with
various forms of familial hypercholesterolemia. It remains unclear as to why A , although
secreted by many cell types throughout the body, is overwhelmingly deposited in the
brain in AD, with only miniscule amounts of non-fibrillar A deposits observed in some
peripheral tissues (24, 25, 26).
Polymorphism of Apolipoprotein E Influences the Incidence and Neuropathology of
AD
Because the APP mutations explained such a small percentage of familial AD, the
search continued for other genetic loci that could predispose to the premature
development of the disease. The second gene to be specifically implicated in familial AD
was that encoding the cholesterol transport protein, apolipoprotein E (apoE). Using
synthetic A as an immobilized ligand, Strittmatter et al. (27) searched for proteins in
human cerebrospinal fluid that were capable of binding to A and isolated apoE. The fact
that the gene encoding apoE was on chromosome 19q in a region that had previously
been shown by these investigators to be linked to late-onset AD led to the discovery that
the inheritance of one or two 4 alleles of the apoE gene confers both a substantially
higher likelihood and an earlier age of onset of the late-onset form of AD (28), whereas
inheritance of the 2 allele appears to do the opposite (29). Therefore, the normally
occurring polymorphism of the apoE protein represents a major genetic risk factor for the
development of AD. Because many E4 carriers do not develop AD and, conversely, many
AD patients do not carry the E4 allele, apoE4 is a risk factor, not a causative mutation.
The mechanism by which the apoE4 protein predisposes subjects to AD remains
unsettled. The most consistent phenotypic clue to the action of apoE4 is the finding that
AD patients harboring one or two 4 alleles have a significantly higher density of both
plaque and vascular A deposits in their brains than do AD subjects lacking this allele
(30, 31, 32, 33, 34). This well confirmed observation has led to several in vitro studies in
which purified apoE4 (generally in a non-lipidated state and at supraphysiological
concentrations) has produced enhanced aggregation of synthetic A peptides under cellfree conditions (e.g. Refs. 35 and 36). Although these studies suggest a role of the apoE4
protein as a facilitator of A aggregation, an alternate explanation for the phenotypic
effect could be a decreased efficiency of clearance of the peptide from the extracellular
space in the presence of apoE4. Regarding A production, evidence obtained in stably
transfected cells co-secreting A and each of the apoE isoforms at physiological levels
suggests that apoE4 does not lead to increased generation of A (37), in contrast to the
mechanism of the APP missense mutations.
4
Missense Mutations in the presenilin 1 and presenilin 2 Genes Cause Many Cases of
Early-onset Familial AD
The discoveries that the APP and apoE genes were implicated in the early-onset and
late-onset forms of familial AD, respectively, left open the question of which other genes
could explain the large majority of early-onset AD cases. Linkage analysis followed by
positional cloning led to the identification in 1995 of two highly homologous familial AD
genes, currently termed presenilin 1 and presenilin 2 (40, 41, 42). These genes encode
467- and 448-residue polypeptides whose sequences and hydropathy profiles suggest that
they contain 7-9 transmembrane domains. The normal functions of these widely
expressed proteins are not yet known. Accordingly, the mechanism by which missense
mutations, of which 27 have already been reported (25 in presenilin 1 and 2 in presenilin
2), cause early-onset familial AD remains to be elucidated by studies in transfected cell
lines and transgenic animals. However, a major clue to their pathogenic effect has come
from recent analyses of A levels in plasma and the conditioned media of skin fibroblasts
obtained from carriers of mutant presenilin genes. Sensitive enzyme-linked
immunosorbent assays show that A peptides ending at residue 42 (A 42) are selectively
augmented by at least some missense mutations in presenilin 1 and 2, whereas levels of
the major form of A (A 40) are largely unchanged (43). A 42 peptides have been found to
have a markedly enhanced rate of aggregation into amyloid fibrils in vitro, compared to
that of A 40 peptides (44). Moreover, immunocytochemical studies using antibodies
specific to the two A C termini reveal that A 42 peptides are the initially deposited
species in the earliest (``diffuse'') plaques in both AD and Down's syndrome brains (8, 9,
45, 46).
In summary, four genes have been implicated to date in familial forms of AD: three that,
when mutant, cause autosomal dominant forms of the disease ( APP, presenilin 1, and
presenilin 2) and one in which a naturally occurring polymorphism (apoE4) represents a
major genetic risk factor for the development of the disease (Table I). Available evidence
strongly suggests that each of these four genes predisposes to the AD phenotype by
enhancing the production and/or the deposition of A peptides (or in the case of apoE4,
perhaps by decreasing its clearance from tissue).
Table I.
Genetic factors predisposing to Alzheimer's disease
Relationships to the -amyloid phenotype are shown. Additional chromosomal loci exist
but are not yet specifically identified.
Chromosome
21
Gene defect
APP mutations
Age of
onset
A phenotype
50s
Production of total A peptides or of
A 42 peptides
5
19
14
1
ApoE4
polymorphism
presenilin
1 mutations
presenilin
2 mutations
60s and
older
40s and
50s
50s
Density of A plaques and vascular
deposits
Production of A
42
peptides
Production of A
42
peptides
The Expression and Post-translational Processing of APP
Growing evidence implicating extracellular A 42 accumulation and resultant amyloid
plaque formation as early and necessary steps in the pathogenesis of the known forms of
heritable AD has heightened interest in understanding the details of the trafficking and
proteolytic processing of APP (reviewed in Ref. 47). This protein occurs in numerous
different isoforms, which arise from alternative splicing of a single gene. The shortest of
the major isoforms (695 amino acids) is expressed almost exclusively in neurons,
whereas the other two common forms (751 and 770 amino acids, respectively) are
expressed both in neural and non-neural cells. Additional heterogeneity of the APP
polypeptides arises from their complex post-translational modifications, including
sulfation, phosphorylation, and both N- and O-linked glycosylation (e.g. Refs. 48, 49,
50). These modifications occur during the trafficking of the protein through the secretory
pathway. APP is co-translationally translocated into the endoplasmic reticulum via its
signal peptide and then matured during passage through the Golgi by acquiring sulfate,
phosphate, and sugar groups, following which a minor percentage of mature molecules is
transported to the plasma membrane via secretory vesicles (47). At the cell surface, some
APP molecules undergo proteolysis by an unidentified protease designated `` secretase,'' which cleaves between lysine 687 and leucine 688, i.e. between residues
16 and 17 of the A region, releasing the large, soluble ectodomain (referred to as APPs) into the medium (51) (Fig. 1). Alternatively, uncleaved surface APP molecules
can undergo endocytosis via clathrin-coated vesicles, apparently mediated by a YENPTY
signal sequence in the distal cytoplasmic tail (52), following which the full-length
precursor is trafficked to late endosomes and lysosomes for apparent degradation (53, 54)
or is rapidly recycled within early endosomes to the cell surface (55). The latter pathway
has been shown to be a principal site for the two proteolytic cleavages that generate the
A peptide (56). A protease termed `` -secretase'' initiates A generation by cleaving
APP after methionine 671, creating a 99-residue (~12 kDa) membrane-retained Cterminal fragment having residue 1 (aspartate) of A as its N terminus (57), and this
results in the secretion of a truncated APPs molecule, called -APPs, into the medium (58)
(Fig. 1). The 12-kDa fragment may then undergo mechanistically enigmatic `` secretase'' cleavages within the hydrophobic transmembrane domain at either valine
711 or isoleucine 713 that release the 40- or 42-residue A peptides, respectively, into the
medium. It appears that only a minority of all biosynthesized APP molecules undergoes
either the -secretory or the -secretory fate; many full-length precursor molecules
remain inserted into internal membranes, particularly in the Golgi.
6
The diverse metabolic fates of APP just summarized are under complex regulation. For
example, several first messengers, including cholinergic agonists and other
neurotransmitters that can activate the phospholipase C/protein kinase C-dependent
pathway, can enhance -secretase cleavage of APP and the consequent release of -APPs
into the extracellular fluid (e.g. Refs. 59 and 60). The mechanism by which this
enhancement occurs is unclear. It does not involve a direct change in the phosphorylation
state of APP (50) and may involve instead the phosphorylation of -secretase or an
enhancement of the trafficking of Golgi-derived vesicles containing APP to the cell
surface (61), where -secretase is known to be active (62). In addition to the regulated
processing of APP through the -secretory pathway, the amyloidogenic processing of
APP (i.e. - followed by -secretase cleavages) can be enhanced, for example by
increases in intracellular free calcium levels (63).
APP Function and Dysfunction
Although the foregoing summary demonstrates that there has been considerable progress
in delineating factors that regulate the processing of APP, the functional implications of
these varied effects remain unclear. The physiological consequences of the enhanced
secretion of either -APPs or A have not been defined. In particular, a specific receptor
for -APPs is not known, although this major secreted derivative has been shown to
stimulate a mitogen-activated protein kinase-mediated p21ras-dependent signal
transduction cascade in cultured cells (65). It has been surmised from the amino acid
sequence of exon 7 of APP and from studies of its function in cultured cells that this
region acts as a serine protease inhibitor of the Kunitz family. -APPs molecules derived
from APP751 and APP770 (which contain this alternatively spliced exon) have been
shown to inhibit several different serine proteases in vitro (66) and to occur in human
plasma as an inhibitor of factor XIa of the coagulation cascade (67, 68). Other functions
of -APPs and/or cell surface holo- APP that have been postulated on the basis of cell
culture experiments include as a trophic (69) or neuroprotective (70) molecule and as a
mediator of cell-cell (71) and cell-substrate (72, 73) interactions.
With regard to APP function in vivo, a mouse in which both APP alleles have been
deleted appears to develop and reproduce normally and has shown minimal phenotypic
alterations to date (74). One explanation for the largely innocuous phenotype resulting
from knocking out APP may be the fact that it is one member of a conserved gene
family that includes the substantially homologous amyloid precursor-like protein-1 and 2 in mammals (75, 76) as well as related homologues in invertebrates (77, 78).
APP will continue to be the subject of intensive study not only because of its role in AD
pathogenesis but also because it is an example of an integral membrane polypeptide that
is expressed both intracellularly and at the cell surface and can be converted at both of
these sites to several different secreted derivatives. The functional consequences of this
highly complex proteolytic processing are important to elucidate. However, there is
currently no compelling evidence that APP is not functioning in patients with
Alzheimer's disease, even in individuals carrying APP missense mutations. Rather, a
large body of data strongly suggests that APP plays a role in both familial and
7
``sporadic'' AD via its A fragment. Increased production (or decreased clearance) of A ,
particularly of the A 42 form, appears to lead gradually to its aggregation in the
extracellular space of the brain and its microvessels. That this process can occur very
early in the development of AD, probably decades prior to the onset of clinical
symptoms, is supported by the finding that patients with Down's syndrome show many
A 42-containing diffuse plaques as early as age 12 years (9). Such individuals do not
develop ``mature'' A plaques containing dystrophic neurites, activated microglia, and
reactive astrocytes until some two decades later, and the appearance of this surrounding
cytopathology is generally associated with the accrual in the plaques of the more
abundantly produced A 40 peptide (9). The morphological evidence of an association of
aggregated, fibrillar A with local cytotoxicity that has long been known from light and
electron microscopic studies of AD brains is consistent with many in vitro studies during
the last few years that indicate that aggregated but not monomeric A peptides can
reproducibly exert toxicity on neurons (e.g. Refs. 79 and 80), astrocytes (81), microglia
(82), and endothelial cells (83) in culture. However, such in vitro experiments necessarily
employ short-term exposure of cells to supraphysiological doses of synthetic A 1-40,
whereas -amyloid deposition in vivo evolves very slowly from peptides with
heterogeneous N and C termini that are secreted at picomolar to low nanomolar
concentrations by neurons, astrocytes, microglia, and endothelial and smooth muscle
cells in the brain.
A more compelling model of the consequences of excessive A accumulation has come
from the production of a transgenic mouse that markedly overexpresses an AD-linked
mutant form of APP in selected neurons (84). These animals secrete large amounts of
both A 40 and A 42 peptides into the extracellular fluid of brain and begin to accumulate
diffuse and compacted (fibrillar) A deposits after about age 5-6 months. Many of these
amyloid plaques are intimately associated with dystrophic neurites, activated microglia,
and reactive astrocytes. Neurofibrillary tangles have not been described to date, but some
cerebral neurons develop immunoreactivity for phosphorylated tau and neurofilament
proteins, suggesting that they are undergoing AD-like cytoskeletal alterations (85). The
substantial similarity of the neuropathology in this model to that of AD strongly supports
the hypothesis that excessive accumulation of first soluble and then aggregated A 42 and
A 40 peptides can initiate Alzheimer-type neuronal and glial changes. This and other
transgenic mouse models should allow a much finer dissection of the temporal course of
the macromolecular, cellular, and behavioral changes that occur in AD. Most
importantly, these animals can be used for the preclinical evaluation of compounds that
interfere with one or another step in the pathogenic mechanism.
Conclusion
The concept that alterations in several distinct genes (four of which have been identified
to date) can lead by different mechanisms to a chronic imbalance between A production
and clearance that results in aggregation of first the 42-residue and then the 40-residue
peptide into cytotoxic plaques is now supported by multiple lines of evidence. Exactly
how aggregated A and the locally secreted and blood-borne proteins that become
associated with it in plaques (e.g. heparan sulfate proteoglycan (86), 1-antichymotrypsin
8
(87), apolipoprotein E (88), complement components (89, 90), serum amyloid P
component (91), and cytokines (92)) exert toxic effects on surrounding cells is an area of
intensive study. It appears that aggregated but not monomeric A peptides can induce cell
dysfunction and death in vitro by a range of presumably interrelated mechanisms that
include oxidative injury (83, 93), alterations in intracellular calcium homeostasis (38, 39),
and cytoskeletal reorganization (64). Sufficient knowledge of some of the principal
elements of the amyloid-induced cascade has emerged that the process of identifying
small molecules which could inhibit one or another step is now well under way. Of
particular therapeutic interest are attempts to decrease the secretion of A peptides from
neuronal and glial cells, something which may be possible to accomplish prior to
knowing the identities of the requisite proteases. Interfering with the aggregation of A 42
and A 40 peptides or inhibiting the toxicity that these extracellular aggregates produce on
neurons, their processes, and glial cells are also of great therapeutic relevance. Finally,
controlling the specialized inflammatory response that appears to be triggered by
aggregated A (including microglial stimulation, activation of the classical complement
cascade, cytokine release, and reactive astrocytosis) should also prove to be of benefit to
patients with this disease. Given the accelerating pace of progress, there can be little
doubt that further biochemical and pharmacological research should lead to a range of
therapeutic options during the next several years.
FOOTNOTES
*
1
The abbreviations used are: AD, Alzheimer's disease; A , amyloid -protein; APP, amyloid precursor protein.
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