Sci. Aging Knowl. Environ., 22 January 2003
Vol. 2003, Issue 3, p. pe1
[DOI: 10.1126/sageke.2003.3.pe1]


Alzheimer's Disease, Neuropeptides, Neuropeptidase, and Amyloid-{beta} Peptide Metabolism

Takashi Saito, Yoshie Takaki, Nobuhisa Iwata, John Trojanowski, and Takaomi C. Saido

Takashi Saito, Yoshie Takaki, Nobuhisa Iwata, and Takaomi C. Saido are at the Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. E-mail: saido{at} John Trojanowski is at the Center for Neurodegenerative Disease Research Institute on Aging and in the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104-4283, USA.;2003/3/pe1

Key Words: mammalian aging • mutations • aggregates • cell senescence • cell loss • life extension • Alzheimer's disease


If humans lived to be 120 to 140 years of age or more, there is a serious possibility that all would develop Alzheimer's disease (AD) (see "Detangling Alzheimer's Disease") in the later years of the human life-span. This prediction stems from the fact that the incidence of AD increases exponentially after the seventh decade of life, although it is not certain that this trend continues in persons beyond age 100. Thus, epidemiological studies indicate that one out of two people who reach the age of 85 and nine out of 10 people who attain the age of 100 will be affected by AD or AD-type brain pathology (1) (see Honig Case Study). What is the significance of this fact? The answers are obviously not entirely clear, because so few individuals have been studied who have attained these ages. But one interpretation is that AD or AD-type pathology may be an ultimate form of brain aging. Note that here we mean to imply that "brain aging" differs from "general aging" in that mitotic quality-control mechanisms play relatively minor roles, simply because differentiated neurons in the adult brain are primarily postmitotic cells.

Until several centuries ago, most human beings lived for only 50 to 60 years, because of harsh living conditions that led to high infant and early adult mortality. Infectious diseases were rampant and no effective therapies to treat them existed; thus, large numbers of children and adults succumbed to bacterial infections. During this time in history, there was practically no "brain aging" of the sort that we have been studying during the past few decades. Therefore, for human beings, the time period between the ages of 50 to 60 and 80 to 100 years is a very new biological time period in the course of the evolution of life, which has taken hundreds of millions of years. Thus, in our view, understanding the mechanism of the aging-dependent development of AD is, at least in part, equal to understanding this new biological time period, which did not even exist until recently.

This reasoning intimates that some aspects of the biological mechanisms that were originally beneficial in maintaining metabolic homeostasis through negative feedback processes when humans lived to be 50 to 60 years of age could become harmful after such ages have been exceeded. Therefore, an understanding of the mechanisms of brain aging, the ultimate form of which may well be represented by AD, should help scientists to create novel strategies for controlling some aspects of what has, until now, been considered to be normal aging.

In this Perspective, we describe our current experimental evidence-based hypothesis that might associate the metabolic homeostasis of neuropeptide(s) in the brain with the aging-dependent development of sporadic AD (SAD) through alteration in the metabolism of amyloid {beta} peptide (A{beta}). It was established during the 1990s, a decade of phenomenal progress in understanding familial AD (FAD), that A{beta} [which is derived from amyloid precursor protein (APP) via the action of {beta}- and {gamma}-secretase] acts as a potent pathogenic agent in the development of AD. Such conclusions were drawn as a consequence of the identification of FAD-causing gene mutations and analyses of their molecular and pathological phenotypes (2). [We consider the microtubule-associated protein tau to be as important as A{beta} in the development of AD, particularly in clinical terms, now that mutations in the tau gene have been shown to cause tauopathy and neurodegeneration (3), but this topic is beyond the scope of this Perspective.] We also propose here a new approach to the metabolic control of A{beta} concentrations in the brain via the modulation of neuropeptide-receptor systems, for the prevention and treatment of AD. All of the neuropeptide receptors are G protein-coupled receptors (GPCRs), which are the most tractable and relevant pharmaceutical targets in molecular pharmacological terms (4).

Neuropeptides and AD

In 1980, Davies and colleagues (5) discovered that concentrations of the neuropeptide somatostatin were significantly reduced in the brains of AD patients. This observation has been confirmed repeatedly by others, but its pathogenic significance has never been fully resolved. Because concentrations of other neuropeptides, such as vasopressin, neuropeptide Y, substance P, and corticotropin-releasing factor, have also been reported to be reduced in AD brains (6-13), the general interpretation has been that the reduction is likely to be, at least in part, a consequence of the degeneration of neuropeptide-generating neurons in AD brains. This assumption does not, however, exclude the possibility that reduction of a specific neuropeptide or neuropeptides may be causal in the pathological cascade of AD development. Consistent with this alternative hypothesis is the finding that the amounts of some neuropeptides decline upon aging before the onset of AD (14, 15).

Meanwhile, our laboratory has been engaged in an effort to identify the physiologically relevant peptidase responsible for the in vivo degradation of A{beta} in the brain (16-19); as described in the next section, the prime suspect, neprilysin, (see Animation 1) is not only involved in the metabolism of neuropeptide(s) but also is likely to be regulated by neuropeptide(s) as part of the homeostatic mechanisms present in the brain.

Accordingly, what Davies and others discovered in the 1980s (5) and what we have identified during the past few years as the major in vivo A{beta}-degrading enzyme (19) now may be combined to formulate a plausible strategy to elucidate the most fundamental question in AD research: What causes the aging-dependent accumulation of A{beta}, which leads to the development of SAD? Moreover, thoughtful integration of these seemingly disparate findings may lead to the development of a new therapy for AD. Such a therapy would not be expected to have the types of side effects that are anticipated for {beta}- and {gamma}-secretase inhibitors, which may also inhibit the biologically indispensable processing of APP and other important substrates (20-22).

Metabolism of A{beta}: The Current Status of Research on the A{beta}-Degrading Enzymes

Because A{beta} is constantly anabolized and catabolized in the brain, the steady-state concentrations of A{beta} are determined by the dynamic balance between anabolic and catabolic activities (23). In contrast to most cases of dominantly inherited FAD and mouse models of A{beta} amyloidosis, elevation of A{beta} anabolism in the brain before the occurrence of the A{beta} pathology is rarely observed in normal aging. One logical presumption, then, is that A{beta} deposition in a large number of SAD cases might be caused primarily by its reduced catabolism [see (23) for further details].

During the past few years, several groups have proposed a variety of candidates for the "real" or authentic A{beta}-degrading enzymes [see (24, 25) for reviews]. Fig. 1 describes the current list of seemingly relevant candidates studied using reverse genetics techniques. Among these candidates, neprilysin and endothelin-converting enzymes (ECEs), which both belong to the M13 clan of metallopeptidases, appear to account for 60 to 80% of the total A{beta}-degrading activity in brain tissue [see (24) for the mathematical basis of this assumption]. Because of the differences in their enzymatic and cellular properties, neprilysin and ECEs are likely to play complementary roles in distinct subcellular compartments; the former degrades A{beta} inside secretory vesicles and on the extracellular surface, whereas the latter does so in acidic compartments represented by the trans-Golgi network (24-26). Incidentally, these peptidases share some neuropeptides as common substrates (26) and thus could be grouped on this basis into a particular family of neuropeptidases (Fig. 2).

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Fig. 1. Current list of A{beta}-degrading enzyme candidates studied with reverse genetic techniques. Abbreviations not previously defined are as follows: KI, (gene) knock-in; tPA, tissue-type plasminogen activator; uPA, urokinase. Asterisk indicates no significance if the difference compared to control mice is <10%: *1, see (19); *2, see (26); *3, see (60); *4, see (61); *5, see (62); *6, see (63); *7, see, for instance, (3). The results with mutant presenilin 1-KI mice represent typical pathogenic alterations in the A{beta} levels, as a positive control, leading to accelerated A{beta} accumulation in the brain. The quantification was performed with an identical enzyme-linked immunosorbent assay developed by Suzuki (64). The mice were ~8 to 10 weeks of age. The gender is likely to be male in each study. T.C.S. accepts the responsibility if any of the data are in error.


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Fig. 2. Cleavage sites of several neuropeptides and A{beta} in proteolysis catalyzed by neprilysin and ECE-1. Neprilysin and ECE-1, which both belong to the M13 clan of peptidases, share similar cleavage site specificity with respect to their substrates (16, 26, 29, 31, 65).

In any case, at the present time, neprilysin (see Animation 1 ) is the most potent A{beta}-degrading enzyme identified in vivo. It is noteworthy that even a heterozygous deficiency of neprilysin leads to an approximately 1.5-fold increase in A{beta} concentrations in brain tissues, a phenotype comparable to those of the FAD-causing gene mutations. Assuming that the parallel with FAD is relevant [see figure 2.8 of (25)], this finding suggests that if neprilysin activity is reduced in human brains to ~50% at the age of 50, the affected individuals will develop AD in 30 to 60 years after this time. Consistent with this hypothesis, the McGeer group has demonstrated that, compared to control mRNAs, neprilysin mRNA concentrations in SAD patients, at relatively early stages of the disease process (Braak Stage II), are significantly and selectively reduced in the hippocampus (a part of the limbic system located in the medial temporal lobe and important in memory formation); the midtemporal gyrus (a convolution or ridge on the surface of the cerebrum); and, to a lesser extent, the cerebellum (the part of the brain that lies just below the posterior part of the cerebrum and plays an essential role in coordinating voluntary movement, controlling muscle tone, and maintaining balance). The fact that the amounts of control neuronal mRNAs that encode cyclophilin and microtubule-associated protein 2 were not changed in tissues from SAD patients indicates that down-regulation of neprilysin expression is not simply a consequence of neurodegeneration (27, 28).

Neprilysin, Neuropeptides, and Aging

Enkephalin is a five-amino acid neuropeptide that is primarily involved in analgesia (pain regulation). Neprilysin was originally identified biochemically as an enkephalin-degrading enzyme in a test tube-based experimental paradigm and was once termed "enkephalinase" (29-31). However, because enkephalin concentrations remain essentially unchanged in the brains of neprilysin-knockout (-KO) mice (32), neprilysin does not appear to be the major rate-limiting enkephalin-degrading enzyme in the brain as a whole. These observations illuminate the danger of depending too much on in vitro assays to predict the in vivo functions of a given molecule, an important caveat in AD research. On the basis of a collection of in vivo and in vitro experiments, we predict that relatively small peptides that can be degraded by exopeptidases (such as aminopeptidases, dipeptidyl peptidases, tripeptidyl peptidases, carboxy peptidases, and peptidyl dipeptidases) probably do not require neprilysin activity for their catabolism as long as they are accessible to these peptidases. Because neprilysin is incapable of degrading peptides larger than ~5 kD (~50 amino acids) (29-31), peptides composed of ~10 to 40 amino acid residues are likely to be relevant substrates. However, the secondary and tertiary structures of the substrates also need to be taken into account, because the three-dimensional structure of neprilysin indicates that only substrates that fit into the active site of the enzyme can be proteolyzed efficiently (33). Indeed, an opioid peptide five amino acids in length was shown to be elevated in a limited region of the hippocampal formation of neprilysin-KO mice (34), contrary to the results of the bulk biochemical quantification (32). This finding indicates that the relative function of neprilysin depends on whether it colocalizes with its substrate and on whether other redundant enzymes exist in the area (34).

Other candidate substrate neuropeptides for neprilysin include somatostatin and substance P, both of which are decreased in AD brains (5, 6). These peptides are good substrates for neprilysin-catalyzed proteolysis in a test-tube paradigm, and substance P has been shown to be metabolically regulated by neprilysin in the colon under inflamatory conditions in vivo (35). Incidentally, insulin-degrading enzyme (IDE), which has been most strongly implicated by some groups (23), has now been shown to play a relatively minor role in A{beta} catabolism (see Fig. 1).

It is of interest that some substrates are known to regulate the activity and expression of neprilysin. Morphine, a mimic of opioid peptides, increases neprilysin enzyme activity in neutrophils in vivo (36), whereas substance P induces neprilysin mRNA expression in fibroblasts in vitro (37) (see Animation 2). In both cases, the receptors involved are likely to be GPCRs (4). Such mechanisms are beneficial in maintaining neuropeptide homeostasis through the formation of negative feedback systems; excessively large amounts of substrate ligands would be reduced by increased neprilysin activity, whereas very small amounts of ligands would have longer life-spans due to the reduced catabolic activity. We thus hypothesize that there may also exist a similar mechanism in the brain (see Animation 3), although we do not yet know the exact identity of the ligand-receptor system. There are several other reasons to predict the presence of such a mechanism in the brain besides the observations outlined above. These can be summarized as follows: (i) neprilysin expression in the hippocampus and neocortex (a portion of the cerebral cortex that has distinct territories involved in sensory, motor, and association functions) is closely associated with interneurons (inhibitory neurons), which are the major sources of neuropeptides (38); (ii) interneurons possess auto- and heteroreceptors (38) for various ligands by which they regulate the homeostatic levels of the ligands via a modulation of the associated signal transduction mechanisms (38); (iii) neprilysin is present inside secretory vesicles and at presynaptic terminals (39, 40) and could thus modulate the intra- and intercellular signals generated by these peptidic ligands, which are produced in synaptic vesicles and released from presynapses; and (iv) the mechanism proposed above can account for the aging-dependent decline of certain neuropeptides and of neprilysin.

We have reproducibly observed an aging-dependent selective reduction of neprilysin activity and expression in the hippocampus and neocortex (25, 39, 40). For these experiments, neprilysin-KO mice were used as a negative control. In the hippocampus (Fig. 3), a prominent local neprilysin reduction was detected in the polymorphic cell layer, the outer molecular layer, and the inner molecular layer of the dentate gyrus, a region of the hippocampal formation involved in the relay of information from the entorhinal cortex to other areas of the hippocampus primarily represented by the CA3 sector. Local neprilysin reduction was also detected in the striatum lucidem (a subcortical mass that has an important excitatory or inhibitory role in processing cortical signals) of the CA3 sector. The CA3 sector processes information coming in through the perforant path (Fig. 3) and out to hippocampal CA sectors 1 to 3 of the ipsilateral and contralateral sides of the brain (40) (see Animation 4). These results indicate that aging causes a local elevation of A{beta} concentrations at the presynapses in these hippocampal areas. Anatomically speaking, these areas correspond to the presynaptic terminal zones of the perforant path and mossy fibers (Fig. 3) originally projecting from the entorhinal cortex [see figure 6.9 of (24) for a schematic], where the initial neurodegeneration takes place in the early stages of the onset and progression of AD (41). The observed decrease in neprilysin expression is not due to a loss of neurons or of presynapses, because presynaptic markers remain unchanged upon aging.

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Fig. 3. Diagram of the physiological localization and aging-associated reduction of neprilysin in the hippocampal formation and entorhinal cortex. Previously described quantitative immunofluorescence observations (24, 39, 40) are summarized in the figure. See also Animation 4. Abbreviations: Or, stratum oriens; Py, stratum pyramidale; Ra, stratum radiata; Lm, stratum lacunosum-moleculare; Mo, molecular layer; Gr, granule cell layer; Po, polymorphic cell layer; Lu, stratum lucidum.

It is not, however, so easy to prove or disprove the mechanism shown in Animation 3 using in vitro paradigms, for the following reasons. Neprilysin is expressed mainly at the presynaptic membranes of interneurons in the brain (39). Although the number of interneurons is smaller than that of excitatory neurons in the hippocampus and neocortex, the number of presynapses formed by interneurons is 100 to 1000 times greater than the number of presynapses formed by excitatory neurons (38, 42). Unless it becomes possible to culture interneurons in a manner that would allow the formation of as many presynapses as occurs in vivo, it will be difficult to analyze the hypothetical ligand-regulated neprilysin activity and expression mechanism in an in vitro paradigm. The most relevant as well as realistic approach would be to quantify neprilysin activity and A{beta} levels in the brains of genetically manipulated mice deficient in a candidate neuropeptide precursor, although this would take more time than the shorter-term in vitro approaches.

Hypothesis: The Mechanism of Aging-Dependent A{beta} Deposition Associated with Neuropeptide Metabolism in the Brain

On the basis of the presumption that the mechanism schematized in Animation 3 also exists in the brain, as it does in some other tissues, we suggest a mechanism for the aging-dependent accumulation of A{beta} in human brains. If we suppose that aging causes a decrease in the synthesis of a neuropeptide (14, 43) that happens to be a ligand involved in the regulation of neprilysin activity and expression, as well as a selective substrate for neprilysin in vivo, then the activity and expression of neprilysin will decrease in a negative feedback manner in order to maintain the homeostasis of the neuropeptide concentrations (see Animation 5). Such a mechanism would probably be beneficial to the brain in the short term, because it would compensate for the reduced synthesis of the ligand peptide. This would have been true particularly in the times when most human beings lived for only 50 to 60 years.

An apparent adverse side effect of this negative feedback mechanism is an increase in A{beta} concentrations, particularly at presynapses, as demonstrated by reverse genetics experiments using neprilysin-KO mice (19, 39). If A{beta} concentrations remain elevated long enough to cause presynaptic dysfunction or degeneration, as demonstrated by a number of studies using human tissues and mouse models that overexpress APP (44, 45), this side effect would positively feed back on itself in a manner that would further lower the neuropeptide levels, because of a reduction in the ability of neurons to synthesize neuropeptides. Such a process would result in a further elevation of the concentration of A{beta}. The presumed presence of such a positive feedback-based, vicious cycle is mathematically consistent with the fact that the accumulation of A{beta} in the brain takes place very slowly in the initial presymptomatic stages of AD, but then increases gradually, and finally escalates catastrophically in what might even correspond to an essentially exponential process (23).

Further, two other positive-feedback vicious cycles likely to contribute to A{beta} accumulation have been described (see Animation 5). One involves an increased synthesis of APP, as observed by some investigators in AD brains (28). This increase is presumably associated with the neuroprotective functions of the soluble form of APP (28) in response to the pathological conditions present. A second process involves an increase in the expression of {beta}-secretase (also known as {beta}-site APP-cleaving enzyme 1, or BACE 1) (as observed in the frontal cortex of AD patients) (46-48), indicating that the increase in BACE 1 occurs at a relatively late stage in the cascade of AD development, according to the different pathological stages of AD development established by Braak and Braak (49). See Animation 6 for the generation of A{beta} by BACE 1 ({beta}-secretase) and the presenilin complex ({gamma}-secretase).

Although none of these cyclic processes are likely to have had any apparent pathogenic effects during past epochs of the human race, when most people died before the age of 60, taken together with an increasing life-span they could synergistically result in the progressive accumulation of A{beta} in the brain and thereby contribute (in conjugation with other genetic and epigenetic risk factors) to an increased propensity to develop AD. This is essentially true of countries where the average life-span has approached or is approaching 80 years. Although this hypothesis is provocative, no published studies on AD or AD-related topics support or contradict it. However, it provides a logical and relevant explanation for the accumulation of A{beta} with advancing age leading to the development of SAD, a disorder in which aging is by far the strongest risk factor.

A New Strategy to Control Brain A{beta} Concentrations Using GPCR Ligand(s)

Although we have not yet identified the ligand-receptor system involved in the maintenance of neprilysin expression and activity schematized in Animation 3, somatostatin is a strong candidate ligand for the following reasons: (i) somatostatin precursor is highly expressed in the hippocampus and neocortex (43); (ii) somatostatin has been shown, in a very reproducible manner, to be decreased in the brains of AD patients; (iii) the somatostatin receptor subtype 4 is exclusively expressed in the hippocampus and neocortex; (iv) somatostatin is an extremely good substrate for neprilysin-catalyzed proteolysis (50); (v) even in primary cultured neurons, and despite the difficulty pointed out in the fourth section of this Perspective, somatostatin seems to up-regulate neprilysin activity to some extent (51); (vi) somatostatin has been shown to improve long-term potentiation, a principal measure of memory, in hippocampal slices (52); and (vii) unlike in neutrophils, treatment of mice with morphine does not have any effect on neprilysin activity or A{beta} levels in the brain (53), thus excluding the opioid peptides as candidate ligands.

As indicated above, the type 4 somatostatin receptor subtype is exclusively expressed in the hippocampus and neocortex (54). Therefore, if somatostatin is one of the major ligands in the neprilysin maintenance system, then a nonpeptidic agonist specific for the type 4 somatostatin receptor would selectively and specifically mobilize the ligand-receptor system only in these brain regions and thus should yield minimal systemic side effects (see Animation 7). This strategy has three major advantages: (i) increased A{beta} degradation; (ii) enhanced memory (through supplementing a factor that is beneficial to memory formation and, at the same time, lacking in AD brains); and (iii) possible activation of {alpha}-secretase(s), including the ADAM proteins (which are disintegrins and metalloproteases) (see Animation 6), as has been demonstrated in cardiac tissues under normal conditions (55). Activation of {alpha}-secretase(s) in the brain would lead to a reduction in the synthesis of A{beta} by reducing the relative involvement of {beta}-secretase in APP processing (56) (see Animation 6).

The advantages of using neprilysin activity to modulate A{beta} concentrations in the brain include the following: (i) neprilysin does not influence the processing of APP and other secretase substrates; (ii) neprilysin has similar Michaelis constant values for all of its substrates and thus preferentially proteolyzes a substrate in large abundance, for example, A{beta} in the brains of patients with AD and with mild cognitive impairment (MCI), a precursor to AD (17); (iii) neprilysin degrades both extracellular and cell-associated A{beta} (57); (iv) neprilysin is a constitutively active enzyme and thus does not require activation of an inactive precursor; (v) unlike matrix metalloproteinases and plasmin, neprilysin does not degrade matrix proteins and thus would not be destructive to structural proteins (29-31); and (vi) neprilysin degrades inflammation-associated peptides, such as substance P, and neurokinins 1 and 2 (29-31) and thus may attenuate neuroinflammation (see McGeer Review). These properties make the use of neprilysin activity for the prevention and therapy of AD complementary to the present mainstream approaches represented by {beta}- and {gamma}-secretase inhibitors and A{beta} vaccination (20-22, 58).

Future Perspectives

We predict that some of the present and future anti-A{beta} approaches to treat AD, many of which are based on different strategies, will be optimally combined in a manner similar to that of the "cocktail therapy" used to treat acquired immunodeficiency syndrome (AIDS) (59). In this latter treatment protocol, used worldwide, a cocktail of three different drugs suppresses disease development, whereas the use of one or two of the three agents generally fails to be effective. Moreover, combining anti-A{beta} strategies with other strategies, such as those that target inflammation, oxidative stress, gonadal steroid deficiencies, cholesterol mismetabolism, tauopathies, etc., will make future prevention and therapy options for AD even more promising.

We are optimistic that the efforts of the AD research community will eventually make it possible for A{beta} concentrations in human brains to be controlled. If this can be accomplished in the early stages of AD development, before massive neurodegeneration takes place, it will serve as a postsymptomatic form of therapy. If it becomes possible to prediagnose the MCI prodromal phase of AD, then presymptomatic intervention can be initiated. The conversion of what has been interpreted as "normal aging" to AD via MCI appears to be a continuous process caused by the gradually accelerating accumulation of several brain pathologies, among which lesions formed by A{beta} are prominent. Thus, it may even be possible to partially control certain aspects of brain aging by maintaining low A{beta} concentrations throughout our lives.

January 22, 2003
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  66. Supported by grants from RIKEN BSI, Ministry of Education, Culture, Sports, Science and Technology, and Ministry of Health, Labor, and Welfare of Japan, as well as from the National Institutes of Health (National Institute on Aging) and the Alzheimer's Association, and by a personal donation from Shigeru Sawada, whose mother died of Alzheimer's disease.
Citation: T. Saito, Y. Takaki, N. Iwata, J. Trojanowski, T. C. Saido, Alzheimer's Disease, Neuropeptides, Neuropeptidase, and Amyloid-{beta} Peptide Metabolism. Science's SAGE KE (22 January 2003),;2003/3/pe1

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