The amyloid precursor protein of Alzheimer disease in human brain ...

The amyloid precursor protein of Alzheimer disease in human brain and blood Qiao-Xin Li, Stephanie J. Fuller, Konrad Beyreuther,* and Colin L. Masters Department of Pathology, The University of Melbourne, Parkville; The Mental Health Research Institute of Victoria, Royal Park Hospital, Parkville, Victoria, Australia; and *Center for Molecular Biology, University of Heidelberg, Germany

Abstract: Studies of the metabolism and function of the amyloid precursor protein (APP) and its proteolytic fragment A␤ in cultured cells, transgenic mice, and post-mortem brain tissue have advanced our understanding of Alzheimer disease (AD). However, the molecular pathogenesis of the disease is still not clear, and we are a long way from finding a cure for the disease. Studies carried out on human platelets and leukocytes have also helped shed light on APP and A␤ metabolism and function. Platelet and leukocyte APP isoforms are processed using mechanisms similar to those in neuronal cells to generate A␤ and soluble forms of APP. The activation of platelets and leukocytes leads to the secretion of APP and A␤, resulting in higher levels of these proteins in serum. APP and A␤ in the circulation may be involved in the regulation of platelet function and in the modulation of immune responses. Because human platelets and lymphocytes produce all forms of APP and secrete amyloidogenic A␤ peptides, these tissues may be useful in monitoring responses to therapeutic interventions directed at APP metabolism. Although not of neuronal origin, further studies on the more accessible ex vivo tissues, including platelets and leukocytes and other blood components, may reveal potential peripheral markers for AD and will further our understanding of the molecular pathogenesis of AD. J. Leukoc. Biol. 66: 567–574; 1999. Key Words: A␤ · platelet · lymphocyte · plasma · neuron

INTRODUCTION Alzheimer disease (AD) is the most common cause of progressive cognitive decline in the aging human population. The main pathological lesions of AD consist of the extracellular deposits of amyloid in the brain in the form of plaques and congophilic angiopathy, as well as intracellular neurofibrillary tangles [1, 2]. The amyloid consists mostly of the self-aggregating A␤ and the smaller p3 peptides, both of which are proteolytically derived from a family of much larger transmembrane glycoproteins, the amyloid precursor proteins (APP) [3]. The A␤ region spans 40–43 residues and is located in the juxta-membrane domain of APP (Fig. 1). Under normal conditions, approxi-

mately 90% of secreted A␤ consists of A␤(1–40) peptide, and about 10% consists of longer A␤(1–42/43) peptides (Fig. 1). Although the A␤(1–42/43) peptides are minor A␤ products, these longer A␤ peptides are more amyloidogenic than the shorter peptides, and are thought to initiate A␤ deposition and plaque formation [4]. In fact, immunocytochemistry studies have shown that A␤(1–42/43) is selectively deposited in all types of amyloid plaques [5, 6], and that the p3(17–42) peptide is a component of the diffuse amyloid deposits in AD [7]. Proteins closely related to APP have been discovered, and are known as the amyloid precursor-like proteins APLP1 and APLP2; however, these do not contain the amyloidogenic A␤ sequence [8, 9]. In this review, we will discuss briefly the findings derived from cultured cell, animal model, and postmortem tissue studies, findings that have been extensively reviewed elsewhere [10, 11], and then we will discuss in greater detail the findings of studies carried out on platelets, lymphocytes, and other blood components.

THE GENETICS OF AD The molecular mechanisms involved in the neuronal degeneration and the progression of dementia in AD are still unclear. The precise role of the A␤ molecule in the pathogenesis of the disease is also unclear; however, one hypothesis is that the development of AD neuropathology is mostly due to the toxic properties of aggregated A␤ fibrils. Some of the evidence that supports the theory that A␤ deposition is involved in the pathogenesis of AD has come from genetic and cell biology studies of the early-onset familial forms of AD (EO-FAD). These studies have identified missense mutations occurring in the APP gene near or within the A␤ sequence in a minority of EO-FAD cases (Fig. 1). However, most mutations that lead to EO-FAD have been found in the genes for the newly identified multi-transmembrane proteins presenilin 1 and 2 [see review in ref. 10]. Despite these inherited forms of AD being linked to mutations in three different proteins, aberrant A␤ metabolism has been found to be associated with all the mutations characterized so far [10, 12], leading to increases in the secretion of all A␤ forms and/or to increases in the ratio of

Correspondence: Colin L Masters, M.D., Department of Pathology, The University of Melbourne, Parkville, Victoria, 3052. E-mail: [email protected] Received March 16, 1999; revised June 16, 1999; accepted June 21, 1999.

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Fig. 1. Structure of the amyloid protein precursor (APP770). Commonly spliced exons—KPI: Kunitz protease inhibitor domain, OX-2: exon with homology to OX-2 domain, and exon 15. SP: signal peptide; CHO: carbohydrate moiety. The shaded areas (TM) represent transmembrane domains. A4CT: membrane-bound fragment of APP after ␤-secretase cleavage; p3CT: membrane-bound fragment after ␣-secretase cleavage. Arrows above the A␤ sequence demonstrate APP mutations that lead to familial early-onset AD.

A␤(1–42/43)/A␤(1–40). Aberrant A␤ metabolism is consistently detected in the studies of the plasma of AD mutation carriers and the media conditioned by the fibroblasts of human carriers [13], as well as in studies of the brains of transgenic mice carrying some of these AD-mutations [14–17]. These studies have consolidated the theory that A␤ deposition is involved in the pathogenesis of AD because the only common factor known to link these different mutations in different proteins is a change in A␤ metabolism. The relationships between these proteins have not been fully characterized; however, recent studies have shown that APP and the presenilins interact closely [18, 19] and that the presenilins may in fact be components of the ␥-secretase complex [20, 21]. Despite these recent advances in the studies of the genetics of early-onset forms of AD, the vast majority of AD cases are sporadic. Several factors (such as estrogen, anti-inflammatory drugs, cerebrovascular disease, oxidants, and metal ions) have been implicated in the pathogenesis of sporadic AD; however, the processes by which these factors may influence disease development in the sporadic cases are still poorly understood. Studies of the pathogenic mutations that lead to EO-FAD are helping to characterize the molecular mechanisms underlying the pathogenesis of the sporadic forms of AD. The only major risk factor that has been linked to an increased likelihood of developing AD has been shown to be the inheritance of one or two apolipoprotein E4 (apoE4) alleles. The possession of apoE4 alleles has also been linked to an earlier age of disease onset. However, it should be emphasized that possession of apoE4 alleles is only a risk factor because not all apoE4 carriers will develop AD [22].

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APP STRUCTURE AND FUNCTION The APP family of glycoproteins consists of at least 10 isoforms that are generated by alternative mRNA splicing, principally of exons 7, 8, and 15, of the APP gene on chromosome 21. The APP isoforms are frequently divided into Kunitz-type protease inhibitor domain-containing isoform (KPI⫹) or Kunitz-type protease inhibitor domain-lacking isoform (KPI⫺), which are generated by the alternative splicing of exon 7. Under normal conditions, the APP lacking the KPI and OX-2 regions (APP695, APP-KPI⫺) is the predominant form produced by neurons (⬃10% of the brain cells are neurons) and neuronal cell lines. The APP-KPI⫹ isoforms (including APP751 and APP770) [23] are the predominant forms expressed in nonneuronal cells under normal conditions; such cells include glia, platelets, and leukocytes, and many non-neuronal cell lines [24–26]. Changes in the APP-KPI⫹/APP-KPI⫺ ratio may occur under pathological conditions because it has been found that a greater proportion of the APP in AD brain consists of KPI⫹ isoforms [27]. The KPI domain may function in an autoregulatory manner by controlling proteolytic events near the cell membrane. The APP-KPI⫹ isoforms may also be more amyloidogenic because overexpression of APP751 in cultured cells results in increased A␤ peptide secretion and decreased p3 peptide secretion [28]. Splicing out exon 15 generates a series of APP molecules (L-APP) that were first discovered in cells of the lymphocyte/ monocyte lineage: T-lymphocytes, macrophages, and microglial cells all express L-APP [29]. The exclusion of exon 15 creates an attachment site for chondroitin sulfate proteoglycans, and

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the binding of a glycosaminoglycan chain close to the A␤ sequence of APP may affect the processing of APP and the production of A␤ or p3 peptides [30]. Resting human T lymphocytes express ⬃40% of APP as L-APP and short-term phytohemagglutinin (PHA)-stimulated lymphocytes express ⬃20% of APP as L-APP. L-APP isoforms are not expressed by neurons, however, they are expressed by non-neuronal cells including glial cells within the central nervous system (CNS) [31]. Between 25 (skeletal muscle) and 70% (aorta, pancreas) of total APP transcripts are L-APP mRNA isoforms. A␤ production appears to occur in all cells and tissues of the body, however, the clinically important A␤ depositions occur only in the brain. This suggests that other factors specific to the CNS may be involved in promoting the A␤ deposition and/or preventing its clearance. Because no overt phenotype has been found in APP-/- knockout mice [32], it is difficult to know what is the precise in vivo function of APP; however, in vitro characterization studies have suggested several functions for APP. APP contains a globular domain (containing heparin-, zinc-, and copper-binding domains), an acidic domain, a KPI domain, and a glycosylation domain that may be involved in dimerization [33]. The cytoplasmic carboxyl-terminal domain of APP contains transduction and internalization signals [34]. APP expressed on the cell surface may serve as a receptor involved in cell-cell or cell-matrix interactions because APP can bind to sulfated proteoglycans, laminin, collagen, or to integrin-like receptors [33, 35–37]. APP has also been shown to be an antagonist for integrin [38]. In addition, APP has growth-promoting and cell-adhesive properties [39–42]. APP interacts with the G0 protein and the brain proteins Fe65 and X11, implicating a role for APP in signal transduction [43–45]. APP can protect neurons against excitotoxic or ischemic insults by stabilizing intracellular calcium [46]. Secreted APP products (sAPP) have been shown to modulate axon growth as well as dendrite branching and dendrite numbers [47]. In neurons, APP is present in axon terminals [48] and has been shown to undergo fast anterograde transport and full transcytosis from the axonal to the somatodendritic compartment [49].

APP PROCESSING AND A␤ PRODUCTION The biogenesis, maturation, and degradation of APP occur via several cellular trafficking and processing pathways. Cleavage within the A␤ sequence of APP by a putative APP ␣-secretase generates soluble amino-terminal fragments of 100–130 kDa (sAPP␣) and an ⬃10-kDa membrane-associated carboxylterminal fragment (p3CT), which can subsequently be cleaved by ␥-secretase to generate the p3 peptides A␤(17–40) and A␤(17–42/43) [50, 51]. Cleavage at the ‘‘␣-secretase’’ site can occur either at the cell surface or within the cell [34, 52]. Electrical depolarization of neurons [53] or activation of protein kinase C by stimulation of various cell surface receptors [54] enhances the ␣-secretase cleavage and results in increased sAPP secretion. An alternative pathway involves cleavage of APP at the amino terminus of A␤ by a protease known as ␤-secretase, yielding sAPP␤ and the A4CT fragment (which extends from the A␤ amino terminus to the carboxyl terminus of

APP (see Fig. 1). Subsequent cleavage of A4CT within its transmembrane region by ␥-secretase generates the A␤(1–40) and A␤(1–42/43). These A␤ peptides are secreted by cells and can be detected in plasma and cerebrospinal fluid [55–58]. The A␤-producing event probably occurs in the early endosomal compartment, the late Golgi compartment, and/or in the endoplasmic reticulum [59, 60]. In non-neuronal cells such as fibroblasts, the ␣-secretase cleavage is the dominant APP processing pathway, whereas in cultured hippocampal neurons and neuronal cell lines, a higher portion of APP is processed by the ‘‘␤/␥-secretases’’ pathway generating relatively greater amounts of A␤ [61]. Although in vitro studies have implicated several proteases in APP processing [62, 63], the in vivo APP ␣-, ␤-, and ␥-secretases are still unknown. Because the APP ␥-secretase cleavage site is thought to be embedded in the lipid bilayer, the mechanism of this proteolytic activity is not understood. Nevertheless, characterizing this proteolytic activity is of great importance because the production of an increased proportion of the more amyloidogenic A␤(x–42/43) by this protease appears to increase the rate of amyloid deposition [4, 5]. The interactions between APP and other molecules of the APP superfamily (APLP1/2) and the presenilin proteins may influence the topology and stability of APP, and may affect the balance between the different APP processing pathways.

PLATELET APP AND A␤ AND THEIR POSSIBLE FUNCTIONS Because platelets are a readily accessible source of human tissue, we have utilized platelets in many of our studies of APP processing and function. Platelets have the highest APP levels of all peripheral tissues, these levels being comparable to the total APP levels in the brain [64]. Platelet APP consists mostly of the sAPP␣-KPI⫹ isoforms (APP770 and APP751). Using APP-specific antibodies, we have shown that the main APP isoforms released by platelets do not include any L-APP isoforms or APLP proteins [65]. Platelets are the primary source of APP in the circulation, producing greater than 90% of the circulating APP [66]. Platelet APP is stored in ␣-granules, as demonstrated by immuno-electron microscopy [25] and sucrose density gradient centrifugation [67]. Approximately 10% of platelet APP is associated with the membrane, and this pool of APP can be separated under alkaline conditions into two types: full-length APP (APPFL-KPI⫹) and carboxyl-terminal truncated membrane-associated APP (APPMem-KPI⫹) [25]. Activation of platelets by thrombin increases the surface expression of APP up to threefold, suggesting that APP-KPI⫹ may regulate hemostatic protease inhibitory activity on the platelet surface [25]. Full-length APP is proteolytically cleaved by a calciumdependent cysteine protease during platelet activation [68]. Platelets have also been found to produce low levels of most other APP metabolites that have been detected in the brain and cell culture, including the amyloidogenic fragment A4CT, the soluble sAPP␤ isoform, and the A␤ peptide [56, 69]. Platelets produce up to 90% of the A␤ in circulation [56, 70] and produce mainly the A␤(1–40) peptide. All these data indicate

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that platelets can process APP by the ␣-, ␤-, and ␥-secretase activities. In contrast to cells of neuronal origin that have a dominant APP ␤-secretase processing pathway, platelets, like other non-neuronal cells, appear to have a dominant ␣-secretase pathway because platelet sAPP␣ levels are much higher than platelet A␤ levels [69]. The sAPP (sAPP␣, sAPP␤) and A␤ can be released by agents that induce platelet degranulation, including the physiological agonists thrombin and collagen, or nonphysiological agonists such as the calcium ionophore A23187 or ionomycin [56, 64, 68, 71]. The released sAPP isoforms potently inhibit coagulation factor XIa and IXa [72, 73], suggesting a role in hemostasis. APP inhibits the intrinsic coagulation factor XIa with a Ki of 450 ⫾ 50 pM and the inhibition can be potentiated 15-fold in the presence of heparin. Zinc also increases the inhibitory activity of sAPP␣ toward factor XIa [74]. Our work shows that recombinant sAPP inhibits platelet aggregation and secretion induced by ADP or adrenaline via the arachidonic acid pathway, indicating that platelet degranulation may result in negative feedback regulation during platelet activation [75]. These data together with the findings that APP possesses growth factor activity [40–42] suggest a physiological function for platelet-derived APP in wound repair and in the microenvironmental regulation of the coagulation cascade. The sAPP inhibition of platelet aggregation appears to be in direct contrast to the A␤ effects on platelet function: A␤ has been shown to augment ADP-dependent platelet aggregation and support platelet adhesion [76, 77]. A␤ also has deleterious effects on the cerebral cortex vessels [78, 79] and peripheral blood vessels [Poliviou, Li, and Khalil, unpublished results] of transgenic mice overexpressing A␤ because it causes loss of vasodilation in response to acetylcholine and increased contractility in response to vasoconstrictors. Therefore a balance in the levels of sAPP␣ and A␤ may be important in hemostasis. A similar balance between APP and A␤ may also be important in the brain because A␤ is thought to be toxic to neurons by disrupting Ca2⫹ homeostasis, whereas sAPP has been shown to protect neurons against excitotoxic insults by stabilizing the intracellular Ca2⫹ concentration [46].

APP AND A␤ IN LYMPHOCYTES It has been shown that lymphocytes, monocytes, neutrophils, macrophages, and microglia all express and secrete APP, with substantial amounts in the form of L-APP-KPI⫹ [26, 80]. PHA-stimulated human T lymphocytes express APP695/751/ 770 in a ratio of 4:7:2, with 20–35% of total APP mRNA consisting of L-APP isoforms in stimulated T lymphocytes [29]. Expression of L-APP in leukocytes and microglial cells may correlate with the dual functions of nonadherence and adherence in the immune system. Microglia in the CNS are functionally akin to macrophages and constitute the main effector cells of the immune response in the CNS. The interaction of microglial APP with different extracellular matrix components has been shown to affect APP secretion as well as intracellular APP metabolism, suggesting that APP plays a role in microglial mobility [81]. A␤ amyloid deposits are infiltrated by microglial cells, suggesting that microglia may also be 570


involved in the AD pathogenic process. APP is also expressed on the cell surface of lymphocytes and monocytes, and the levels of this APP can be increased when the cells are exposed to activation signals such as the mitogenic lectins or antibodies to the antigen receptors of B and T cells [82]. This suggests that APP may act as a cell surface receptor in the immune system. Lymphoblastoid cells from patients with EO-FAD produce higher levels of APP as well as higher levels of intracellular 12to 16-kDa APP fragments (including the A4CT fragment) when compared to normal cells from control subjects. These cells have also been shown to express increased amounts of interleukin-1 and ␣1-antichymotrypsin when compared to control cells [83]. The EO-FAD cells also uniquely express a 68-kDa Ca2⫹-dependent serine protease that binds strongly to APP, and that has been suggested to be a ␤-secretase [84]. Because this protease was only found in familial AD cells, the findings suggest an abnormality of posttranslational regulation of APP in some cases of AD. In one study of familial AD lymphoblastoid cells, a 4-kDa peptide confirmed to be A␤ by sequencing was detected in cell lysates, suggesting that lymphocytes produce intracellular A␤. Secreted A␤ or p3 peptides were not detected in this study; however, the familial AD cells (and not control cells) were found to secrete a 12-kDa peptide that was identified as the p3CT fragment (Fig. 1) [85]. Using metabolic labeling and immunoprecipitation studies, we have now demonstrated that freshly isolated lymphocytes as well as EpsteinBarr virus-transformed lymphocytes derived from both familial AD and control cases can secrete A␤, p3, and possibly two other APP-derived peptides containing portions of the A␤ sequence [Fuller and Masters, unpublished results]. Lymphocytes, therefore, are likely to be another source of the A␤ that is found in blood. According to the calcium hypothesis of brain aging, disturbances of intracellular calcium homeostasis [Ca2⫹]i play a key role in the pathology of AD. In normal T lymphocytes that have been activated with PHA, A␤ has been found to amplify [Ca2⫹]i signaling; however, in the activated lymphocytes from sporadic as well as EO-FAD patients, this sensitivity to A␤ was found to be significantly reduced [86]. This study also found a K⫹ channel dysfunction in AD lymphocytes. Another study of the effects of A␤ on lymphocytes demonstrated that lymphocytes from healthy controls proliferated when stimulated with A␤ peptides, whereas lymphocytes from AD patients did not proliferate [87]. The authors suggested that autoreactive lymphocytes with specificity for metabolic products of APP may occur in healthy individuals, and that this mechanism may be impaired in AD. Another comparison of AD and control T lymphocytes has demonstrated that there is a reduced number of tumor necrosis factor ␣ receptors in AD lymphocytes, which may indicate a systemic immune activation in AD patients [88].

APP AND A␤ IN PLASMA Low concentrations (approximately 10 pM) of carboxylterminally truncated APP-KPI⫹ are found in plasma when blood is carefully collected with minimal platelet activation [89], as compared to 60 pM using normal blood collection techniques [66]. Although the origin of plasma APP is uncerhttp://www.jleukbio.org

tain, our studies suggest that the major source may be platelets due to their high concentration of APP (30 nM) compared to other cells in the circulation. A study of APP levels in the platelets and plasma of a patient with grey platelet syndrome, in which platelets have a characteristically low ␣-granule content, supports the theory that plasma APP is mostly derived from platelets. This study demonstrated a reduction of APP in platelets of the patient, which was associated with a similar reduction in plasma APP levels [25]. As mentioned earlier, platelets may also be the main source of plasma A␤ [70]. Serum contains approximately twofold more APP and A␤ than plasma: this is consistent with the release of APP and A␤ by platelets during blood clotting [56]. The levels of APP and A␤ in plasma are relatively low when compared to platelet levels or CSF levels (approximately 10-fold greater) of these proteins, suggesting that APP and A␤ have short half-lives once secreted into the plasma. In support of this, turnover rates of ⬃2 h for A␤ and ⬃7 h for APP have been observed in the gene-targeted mouse expressing the human A␤ sequence [90]. The A␤ that deposits as amyloid in AD brains is most likely to have been produced locally; it is also possible that some of this A␤ may be derived from the circulation. The mechanism involved in the delivery of A␤ across the blood-brain barrier is not clear, however, it has been suggested that A␤ can be taken into the brain as a complex with HDL3 and VHDL in association with ApoJ [91]. In particular, circulating A␤ may contribute to cerebrovascular amyloid (congophilic angiopathy), one of the pathological features of AD, because it can be internalized and accumulated in human smooth muscle cells [92]. Several studies that have compared platelets derived from AD patients with those derived from normal controls have found differences in platelet membrane structures and APP metabolism. Abnormalities in platelet membrane fluidity (with a 50% increase shown in AD patients) have been demonstrated in most of the studies [see review in ref. 93]. Some groups have reported altered APP processing in platelets of patients with AD compared with controls [94, 95]. Studies of platelets derived from patients with severe AD suggest that this altered APP processing may be, at least in part, the result of abnormalities in these platelets because they appear to be hyperacidified upon thrombin activation [96]. Our studies have identified an elevation in the proportion of the 130-kDa sAPP species in the plasma of patients with moderate to severe dementia of the Alzheimer’s type when compared to agematched controls [97]. All these studies indicate that ADrelated abnormalities of APP metabolism (which can increase the A␤ concentration and result in A␤ deposition) in the CNS might be reflected in the circulation, despite the fact that CNS cells and peripheral cells (including platelets and lymphocytes) have different APP isoforms and different preferred processing pathways.

CONCLUSIONS APP and A␤ are produced by cells of peripheral tissues as well as by cells of the CNS; however, the mechanism by which amyloid deposition occurs only in the brain is not clear. This

may be due to the metabolism of APP by different preferred pathways, the presence of factor(s) that may accelerate or prevent the deposition of A␤, or it may be due to the more effective removal of A␤ from peripheral tissues. Studies of both brain-derived cells as well as non-CNS cells and tissues, including platelets and lymphocytes, are being carried out to determine the differences in the metabolism of the APP and A␤ between CNS and non-CNS tissues. The information drawn from these studies may help to develop a therapeutic strategy for AD, and further studies on platelets and leukocytes and other components of blood may also reveal potential peripheral markers for AD. Because human platelets and lymphocytes produce all forms of APP and secrete amyloidogenic A␤ peptides, these tissues may also prove to be useful in monitoring responses to therapeutic interventions directed at APP metabolism.

ACKNOWLEDGMENTS This work was supported in part by grants from the National Health and Medical Research Council of Australia (no. 950663). Konrad Beyreuther is supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium fu¨r Forschung und Technologie.

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