Molecular Psychiatry (1999) 4, 524–528  1999 Stockton Press All rights reserved 1359–4184/99 $15.00

PROGRESS It all sticks together—the APP-related family of proteins and Alzheimer’s disease TA Bayer1, R Cappai2, CL Masters2, K Beyreuther3 and G Multhaup3

1Department of Psychiatry, University of Bonn Medical Center, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany; 2Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia; 3ZMBH, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany

In the present review, we shall discuss the pros and cons of a possible functional relationship and contribution of the APP family members (APP, APLP1 and APLP2) to the development of Alzheimer’s disease: (1) APP, APLP1 and APLP2 are highly homologous proteins with similar protein domain organization. (2) All APP family proteins have been found to be aggregated in typical Alzheimer’s disease lesions. (3) Several other proteins have been implied to provide a functional link among the APP-related proteins. In normal adult brain APP, APLP1 and APLP2 are involved in synaptic processes important for memory function. We hypothesize that the functional loss of members of the APP family contributes to the gradual cognitive decline in Alzheimer’s disease patients. Keywords: APP; APLP1; APLP2

The neuropathological hallmarks of Alzheimer’s dis- gene has been located at chromosome 19q13.1 and ease (AD) include neurofibrillary tangles, neuritic pla- shown to consist of 17 exons.14 APP, APLP1 and ques, and congophilic angiopathy. While tangles are APLP2 have the structure of type I integral membrane predominantly composed of an abnormally phos- glycoproteins with a major extracellular component phorylated tau protein, ␤-amyloid has been identified and a short cytoplasmatic tail3 (Figure 1), are highly as a major component of both neuritic plaques and blood vessels with amyloid angiopathy in AD. Since the purification and characterization of cerebral amy- loid protein from senile plaques in AD and in older persons with Down syndrome,1,2 the protein compo- nents of plaques have received considerable interest. The ␤-amyloid precipitates are aggregates of 39- to 43- residues ␤A4 peptide,3,4 which originate from a larger ␤-amyloid precursor protein (APP). The discovery of amyloid precursor-like proteins related to APP classi- fies APP as a member of a multigene family.5–8 The highly homologous nature of the proteins raises the possibility of a functional relationship. Wasco et al6 isolated the human APLP2 gene, which has been assigned to chromosome 11 q23–q25.9 The mouse APLP2 promoter lacks a typical TATA box, is GC-rich, and contains several sequences for transcription factor Figure 1 The APP protein family and domain organization 10,11 binding. The mouse APLP1 genomic structure is illustrated. SP denotes the signal peptide domain; CYS the revealed 17 exons and a 5Ј region that contains puta- conserved cysteine-rich domain; Acidic the less conserved tive binding sites for AP-1, heat-shock protein, and Sp1 acidic domain; Glycosylation the N- and O-glycosylation devoid of apparent TATA and CCAAT boxes.12 Human domain; ␤A4 the ␤A4/␤-amyloid domain of APP; and TM APLP1 cDNA has been cloned recently,13 the protein indicates the transmembrane domain. The extracellular was found in human CSF suggesting that soluble domain on the aminoterminal site of the transmembrane APLP1 is released from brain cells. The human APLP1 region reveals highest divergence (arrow). The KPI domain (exon 7), OX-2 domain (exon 8) and exon 15 of APP and KPI domain (homologous to APP exon 7) and a divergent region Correspondence: TA Bayer, PhD, Department of Psychiatry, Uni- of 12 amino acids on the NH2-terminal side of the transmem- versity of Bonn Medical Center, Sigmund-Freud-Strasse 25, brane domain (12-aa exon) of APLP2 are alternatively spliced. 53105 Bonn, Germany. E-mail: bayerȰuni-bonn.de No alternative splice variants have been described for APLP1. Received 2 December 1998; revised 1 March 1999; accepted 8 Note, the ␤A4 domain is absent in APLP1 and APLP2. Not March 1999 drawn to scale. APP and Alzheimer’s disease TA Bayer et al 525 STAR Sequence alignment of the human APP superfamily. The sequences were extracted from the Swissprot data base. Alignment with Megalign (Lasergene, DNA Figure 2 Inc, Madison, WI, USA). Black boxes denote residues that match the Consensus exactly. APP and Alzheimer’s disease TA Bayer et al 526 homologous proteins (Figure 2) evolved from the com- within the first week after birth. The survivors showed mon ancestor APLP1 (Figure 3). The ␤A4/␤-amyloid deficits in balance and/or strength, but appeared heal- domain, however is unique to APP and absent in other thy up to 13 months of age.30 Recently, Rassoulzadegan members of the superfamily. APP is alternatively et al31 investigated a large APLP2 deletion in the gen- spliced (exons 7, 8 and 15), which gives rise to at least omic locus, suggesting a role of APLP2 in mitotic segre- eight isoforms expressed in a cell-type specific manner. gation of the genome and establishment of a proper APLP2 has two alternatively spliced exons, one com- nuclear structure. Increased expression of cell-associa- prises the Kunitz protease inhibitor domain encoding ted APLP2, but not APLP1, was detected in ␤A4- exon and a 12-amino acid long exon situated adjacent treated APP−/− and APP+/+ neuronal cell cultures but 15–17 to the transmembrane domain. The latter not in H2O2-treated cultures. These findings demon- resembles the tissue-specific alternative splicing of strate that ␤A4 toxicity does not require an interaction exon 15 of APP, corresponding to 18 residues and was of the ␤A4 peptide with the parental molecule (APP) first reported in leukocytes.18 APP isoforms termed L- and is therefore distinct from prion protein neurotoxic- APP that lack sequences encoded by exon 15 are modi- ity that is dependent on the expression of the parental fied by chondroitin sulfate glycosaminoglycans (CS- cellular prion protein, and that APLP2 expression may GAG), whereas the APLP2-763 isoform which contains be functionally triggered by ␤A4.32 the 12-amino acids insert is not modified by CS-GAG.19 Induction of apoptosis by trophic factor withdrawal Studies on the protein level and quantitative mRNA upregulated APP and APLP2 protein synthesis, and the analysis revealed an AD-associated increase in the pro- alternative splicing pattern of APP in neuronal PC12 portion of APP exon 7 encoded Kunitz-type serine pro- cells.33 The members of the APP gene family have been tease inhibitory-containing isoforms.20,21 The Kunitz- implicated in neuronal differentiation, since mRNA type serine protease inhibitory sequence (KPI) in the levels increased after retinoic acid induction of human APP gene diverged from its ancestral gene about 270 neuroblastoma cells (SH-SY5Y) of both APLP2 and million years ago and was inserted into the gene soon APP, whereas the increase in APLP1 mRNA expression after duplication.22 No APLP1 spliced transcripts have was significantly higher.34 Phosphorylation-dependent been discovered so far. Detailed alternative splicing regulation of APLP2 in the cytoplasmic domain by pro- patterns of APP and APLP2 have been previously tein kinase C and cdc2 kinase appears to be similar to described and reviewed by Sandbrink et al.23 that of APP. APLP1 has been shown to be phosphoryl- APLP2 is widely expressed in neural and non-neural ated by protein kinase C.35 tissues.6,24,25 In situ hybridization and quantitative The hypothesis of environmental toxins for AD has polymerase chain reaction revealed that APLP2 and received growing attention in the past years. The APP mRNA are expressed in similar, if not identical, identification of a copper (II) binding site in APP and neuronal populations and at similar levels. APLP2 APLP2 has been suggested to be involved in electron appears to mature through the same unusual transfer and radical reactions.36,37 A strong heparin- secretory/cleavage pathway as APP,8 and is found in binding site within a region conserved in rodent and post-synaptic compartments.24,26 Evidence that APP human APP, APLP1 and APLP2, was identified in the and APLP2 have a shared cellular activity besides the carbohydrate domain suggesting a role in cell KPI domain comes from the observation that recombi- adhesion.38 APP and its homologues behave as a family nant human APLP2 expressed in the yeast Pichia pas- of zinc-modulated, heparin-binding proteins.39 toris can stimulate neurite outgrowth.27 Tranfection Additionally, a conserved zinc (II) binding site was experiments of mouse APLP2 cDNA revealed that demonstrated to interact with the heparin-binding APLP2 is modified by CS-GAG at a region with little site.40 homology to APP, suggesting functional regulation, A role of APLP1 and APLP2 in AD has gained further which has been suggested to have a role in axonal interest with the discovery that both proteins are pathfinding and/or synaptogenesis in the olfactory present in typical AD-associated lesions. Crain et al25 bulb and embryonic neurons.19,28,29 APLP1 is increased showed that APLP2 antibodies label dystrophic neur- during cortical synaptic development, suggesting a role ites, a subset of plaques, neurons and reactive astro- in synaptogenesis or synaptic maturation.26 cytes in AD, which was confirmed by others.41,42 More A role in axonal pathfinding has been challenged by recently, our group reported43 that APLP1 protein also Koch et al30 who studied APLP2 knock-out mice, precipitates in a subpopulation of neuritic plaques, a which failed to show impairment of axonal outgrowth finding, which was supported recently.42 of olfactory neurons following bulbectomy. Eighty per Several proteins have been implied to provide a cent of APP and APLP2 double knock-out mice died functional link among the family of APP-related pro-

Figure 3 Phylogenetic tree of APP, APLP1 and APLP2 and ancestral relationships between sequences. Units indicate the number of substitution events. The length of each pair of branches represents the distance between sequence pairs. APP and Alzheimer’s disease TA Bayer et al 527 teins. Duilio et al44 reported that Fe65 and another purification and characterization of a novel cerebrovascular amy- Fe65-like protein (Fe65L2) interact with APP, APLP1 loid protein. Biochem Biophys Res Commun 1984; 120: 885–890. 3 Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzes- and APLP2. Another human homologue of Fe65 desig- chik KH et al. The precursor of Alzheimer’s disease amyloid A4 nated hFe65L was found to bind APP and APLP2, but protein resembles a cell surface receptor. Nature 1987; 325: 733– not APLP1.45 Fe65 has been shown to bind APP, 736. APLP1 and APLP2 via a phosphotyrosine interaction 4 Tanzi RE, Gusella JF, Watkins PC, Bruns GAP, St George-Hyslop PH, Van Keuren ML et al. The amyloid ␤ protein gene: cDNA clon- domain, which binds to the same motif present in the ing, mRNA distribution, and genetic linkage near the Alzheimer conserved cytoplasmic domains. RNA analyses reveal locus. Science 1987; 235: 880–994. that Fe65 is predominantly expressed in brain and in 5 Wasco W, Bupp K, Magendantz M, Gusella JF, Tanzi RE, Solomon the regions most affected by Alzheimer’s disease-asso- F. Identification of a mouse brain cDNA that encodes a protein ciated neuropathology.46 Interestingly, APLP1 binds to related to the Alzheimer disease-associated amyloid beta protein 47 precursor. Proc Natl Acad Sci USA 1992; 89: 10758–10762. prion protein, a membrane protein, which is linked 6 Wasco W, Gurubhagavatula S, Paradis MD, Romano DM, Sisodia to the membrane by a GPI anchor. Interaction between SS, Hyman BT et al. Isolation and characterization of APLP2 enco- prion protein and a member of the APP family raises ding a homologue of the Alzheimer’s associated amyloid beta pro- the possibility that similar mechanisms feature in the tein precursor. Nat Genet 1993; 5: 95–100. 48 7 Sprecher CA, Grant FJ, Grimm G, O’Hara PJ, Norris F, Norris K et pathogenesis of scrapie and AD. al. Molecular cloning of the cDNA for a human amyloid precursor A plethora of information about the members of the protein homolog: evidence for a multigene family. Biochemistry APP-related gene family supports the notion, that all 1993; 32: 4481–4486. proteins participate in the development of AD. (1) APP, 8 Slunt HH, Thinakaran G, Von Koch C, Lo AC, Tanzi RE, Sisodia APLP1 and APLP2 are highly homologous genes, with SS. Expression of a ubiquitous, cross-reactive homologue of the mouse beta-amyloid precursor protein (APP). J Biol Chem 1994; similar protein domain organization (Figure 2). (2) 269: 2637–2644. APP, APLP1 and APLP2 are post-translational modi- 9 von der Kammer H, Loffler C, Hanes J, Klaudiny J, Scheit KH, fied and show similar binding capabilities with differ- Hansmann I. The gene for the amyloid precursor-like protein ent molecules. (3) APP, APLP1 and APLP2 exhibit APLP2 is assigned to human chromosome 11q23–q25. Genomics overlapping cellular expression patterns in normal 1994; 20: 308–311. 10 von Koch CS, Lahiri DK, Mammen AL, Copeland NG, Gilbert DJ, brain. (4) APP, APLP1 and APLP2 exhibit overlapping Jenkins NA et al. The mouse APLP2 gene. Chromosomal localiz- staining patterns in Alzheimer’s disease typical ation and promoter characterization. J Biol Chem 1995; 270: lesions. (5) Increasing evidence accumulates that Fe65 25475–25480. can bind to homologous domains of APP, APLP1 and 11 Yang Y, Martin L, Cuzin F, Mattei MG, Rassoulzadegan M. Gen- omic structure and chromosomal localization of the mouse CDEI- APLP2 (Figure 2). Generation of transgene mice over- binding protein CDEBP (APLP2) gene and promoter sequences. expressing APLP1 or APLP2 are required to further Genomics 1996; 35: 24–29. support the hypothesis of a participation of APLP1 12 Zhong S, Wu K, Black IB, Schaar DG. Characterization of the gen- and/or APLP2 in the molecular pathogenesis of AD. To omic structure of the mouse APLP1 gene. Genomics 1996; 32: date only APP overexpressing mice have been reported 159–162. 49,50 13 Paliga K, Peraus G, Kreger S, Du¨ rrwang U, Hesse L, Multhaup G et to elicit AD-associated neuropathological changes. al. Human amyloid precursor-like protein 1—cDNA cloning, We hypothesize that the APP family has a crucial ectopic expression in COS-7 cells and identification of soluble role in synapse formation and maintenance, synaptic forms in the cerebrospinal fluid. Eur J Biochem 1997; 250: 354–363. plasticity and cellular mechanisms underlying learning 14 Lenkkeri U, Kestila M, Lamerdin J, McCready P, Adamson A, Olsen and memory. These proteins may have a function in A et al. Structure of the human amyloid-precursor-like protein gene APLP1 at 19q13.1. Hum Genet 1998; 102: 192–196. memory consolidation linking synaptic efficacy and 15 Sandbrink R, Masters CL, Beyreuther K. Similar alternative splicing synaptic dynamic processes. Tightly balanced regu- of a non-homologous domain in beta A4-amyloid protein precur- lation and transport of the APP family proteins to sub- sor-like proteins. J Biol Chem 1994; 269: 14227–14234. cellular compartments are required for normal infor- 16 Sandbrink R, Masters CL, Beyreuther K. APP gene family: unique mation processing between single neurons and cortical age-associated changes in splicing of Alzheimer’s betaA4-amyloid protein precursor. Neurobiol Dis 1994; 1: 13–24. areas. In AD brain, misregulation of these proteins 17 Sandbrink R, Masters CL, Beyreuther K. APP gene family. Alterna- could lead in a first step to intraneuronal accumulation tive splicing generates functionally related isoforms. Ann NY Acad resulting in a disruption of transport function of pro- Sci 1996; 777: 281–287. teins and initiation of neurotoxic events. In a second 18 Ko¨nig G, Monning U, Czech C, Prior R, Banati R, Schreiter-Gasser step the appearance of extracellular protein plaques in U et al. Identification and differential expression of a novel alterna- tive splice isoform of the beta A4 amyloid precursor protein (APP) brain parenchyma manifests irreversible neurodegen- mRNA in leukocytes and brain microglial cells. J Biol Chem 1992; eration. Consequently, absence of functional active 267: 10804–10809. APP, APLP1 or APLP2 proteins may cause chronic cog- 19 Thinakaran G, Slunt HH, Sisodia SS. Novel regulation of chondro- nitive decline. itin sulfate glycosaminoglycan modification of amyloid precursor protein and its homologue, APLP2. J Biol Chem 1995; 270: 16522–16525. 20 Moir RD, Lynch T, Bush AI, Whyte S, Henry A, Portbury S et al. References Relative increase in Alzheimer’s disease of soluble forms of cer- ebral abeta amyloid protein precursor containing the kunitz pro- 1 Masters CL, Simms G, Weinmann NA, Multhaup G, McDonald BL, tease inhibitory domain. J Biol Chem 1998; 273: 5013–5019. Beyreuther K. Amyloid plaque core protein in Alzheimer’s disease 21 Johnston JA, Norgren S, Anneren G, Cowburn RF, Lannfelt L. A and Down’s syndrome. Proc Natl Acad Sci USA 1985; 82: 4245– new quantitative solution hybridisation-RNase protection assay for 4249. APP and APLP2 mRNA. Brain Res Mol Brain Res 1996; 43: 77–84. 2 Glenner GG, Wong CW. Alzheimer’s disease: initial report of the 22 Ikeo K, Takahashi K, Gojobori T. Evolutionary origin of a Kunitz- APP and Alzheimer’s disease TA Bayer et al 528 type trypsin inhibitor domain inserted in the amyloid beta precur- 37 Multhaup G, Ruppert T, Schlicksupp A, Hesse L, Bill E, Pipkorn sor protein of Alzheimer’s disease. J Mol Evol 1992; 34: 536–543. R et al. Copper-binding amyloid precursor protein undergoes a site- 23 Sandbrink R, Monning U, Masters CL, Beyreuther K. Expression of specific fragmentation in the reduction of hydrogen peroxide. Bio- the APP gene family in brain cells, brain development and aging. chemistry 1998; 37: 7224–7230. Gerontology 1997; 43: 119–131. 38 Multhaup G. Identification and regulation of the high affinity bind- 24 Thinakaran G, Kitt CA, Roskams AJ, Slunt HH, Masliah E, von ing site of the Alzheimer’s disease amyloid protein precursor (APP) Koch C et al. Distribution of an APP homolog, APLP2, in the mouse to glycosaminoglycans. Biochimie 1994; 76: 304–311. olfactory system: a potential role for APLP2 in axogenesis. J Neuro- 39 Bush AI, Pettingell WJ, de Paradis M, Tanzi RE, Wasco W. The sci 1995; 15: 6314–6326. amyloid beta-protein precursor and its mammalian homologues. 25 Crain BJ, Hu WD, Slunt HH, Koo EH, Price DL, Thinakaran G et Evidence for a zinc-modulated heparin-binding superfamily. J Biol al. Expression and distribution of amyloid precursor protein-like Chem 1994; 269: 26618–26621. protein-2 in Alzheimer’s disease and in normal brain. Am J Pathol 40 Multhaup G, Mechler H, Masters CL. Characterization of the high 1996; 149: 1087–1095. affinity heparin binding site of the Alzheimer’s disease beta A4 26 Kim TW, Wu K, Xu JL, McAuliffe G, Tanzi RE, Wasco W et al. amyloid precursor protein (APP) and its enhancement by zinc(II). Selective localization of amyloid precursor-like protein 1 in the J Mol Recognit 1995; 8: 247–257. cerebral cortex postsynaptic density. Brain Res Mol Brain Res 1995; 41 Bayer TA, Weggen S, Hesse L, Cappai R, Masters CL, Beyreuther 32: 36–44. K et al. Distribution of amyloid precursor-like protein 2 in normal 27 Cappai R, Mok SS, Galatis D, Tucker DF, Henry A, Beyreuther K et and Alzheimer’s disease hippocampal formation. Alzheimer’s Res al. Recombinant human amyloid precursor-like protein 2 (APLP2) 1997; 3: 199–203. expressed in the yeast Pichia pastoris can stimulate neurite out- 42 McNamara MJ, Ruff CT, Wasco W, Tanzi RE, Thinakaran G, Hyman growth. FEBS Lett 1999; 442: 95–98. BT. Immunohistochemical and in situ analysis of amyloid precur- 28 Thinakaran G, Sisodia SS. Amyloid precursor-like protein 2 sor-like protein-1 and amyloid precursor-like protein-2 expression (APLP2) is modified by the addition of chondroitin sulfate glycosa- in Alzheimer disease and aged control brains. Brain Res 1998; 804: minoglycan at a single site. J Biol Chem 1994; 269: 22099–22104. 45–51. 29 Sisodia SS, Thinakaran G, Slunt HH, Kitt CA, Von Koch CS, Reed 43 Bayer TA, Paliga K, Weggen S, Wiestler OD, Beyreuther K, Mul- RR et al. Studies on the metabolism and biological function of thaup G. Amyloid precursor-like protein 1 accumulates in neuritic plaques in Alzheimer’s disease. Acta Neuropathol (Berl) 1997; 94: APLP2. Ann NY Acad Sci 1996; 777: 77–81. 519–524. 30 von Koch CS, Zheng H, Chen H, Trumbauer M, Thinakaran G, van 44 Duilio A, Faraonio R, Minopoli G, Zambrano N, Russo T. Fe65L2: der Ploeg LH et al. Generation of APLP2 KO mice and early post- a new member of the Fe65 protein family interacting with the intra- natal lethality in APLP2/APP double KO mice. Neurobiol Aging cellular domain of the Alzheimer’s beta-amyloid precursor protein. 1997; 18: 661–669. Biochem J 1998; 330: 513–519. 31 Rassoulzadegan M, Yang Y, Cuzin F. APLP2, a member of the Alzh- 45 Guenette SY, Chen J, Jondro PD, Tanzi RE. Association of a novel eimer precursor protein family, is required for correct genomic seg- human FE65-like protein with the cytoplasmic domain of the beta- regation in dividing mouse cells. Embo J 1998; 17: 4647–4656. amyloid precursor protein. Proc Natl Acad Sci USA 1996; 93: 32 White AR, Zheng H, Galatis D, Maher F, Hesse L, Multhaup G et 10832–10837. al. Survival of cultured neurons from amyloid precursor protein 46 Bressler SL, Gray MD, Sopher BL, Hu Q, Hearn MG, Pham DG et knock-out mice against Alzheimer’s amyloid-beta toxicity and oxi- al. cDNA cloning and chromosome mapping of the human Fe65 dative stress. J Neurosci 1998; 18: 6207–6217. gene: interaction of the conserved cytoplasmic domains of the 33 Araki W, Wurtman RJ. Increased expression of amyloid precursor human beta-amyloid precursor protein and its homologues with protein and amyloid precursor-like protein 2 during trophic factor the mouse Fe65 protein. Hum Mol Genet 1996; 5: 1589–1598. withdrawal-induced death of neuronal PC12 cells. Brain Res Mol 47 Yehiely F, Bamborough P, Da CM, Perry BJ, Thinakaran G, Cohen Brain Res 1998; 56: 169–177. FE et al. Identification of candidate proteins binding to prion pro- 34 Beckman M, Iverfeldt K. Increased gene expression of beta-amyloid tein. Neurobiol Dis 1997; 3: 339–355. precursor protein and its homologues APLP1 and APLP2 in human 48 Prusiner SB. Some speculations about prions, amyloid, and Alzhei- neuroblastoma cells in response to retinoic acid. Neurosci Lett mer’s disease. N Engl J Med 1984; 310: 661–663. 1997; 221: 73–76. 49 Sturchler-Pierrat C, Abramowski D, Duke M, Wiederhold KH, Mistl 35 Suzuki T, Ando K, Isohara T, Oishi M, Lim GS, Satoh Y et al. Phos- C, Rothacher S et al. Two amyloid precursor protein transgenic phorylation of Alzheimer beta-amyloid precursor-like proteins. mouse models with Alzheimer disease-like pathology. Proc Natl Biochemistry 1997; 36: 4643–4649. Acad Sci USA 1997; 94: 13287–13292. 36 Multhaup G, Schlicksupp A, Hesse L, Beher D, Ruppert T, Masters 50 Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, CL et al. The amyloid precursor protein of Alzheimer’s disease in Blackwell C et al. Alzheimer-type neuropathology in transgenic the reduction of copper(II) to copper(I). Science 1996; 271: 1406– mice overexpressing V717F beta-amyloid precursor protein. Nature 1409. 1995; 373: 523–527.