REVIEWThe Gene Defects Responsible for Familial Alzheimer's Disease

Neurobiology of Disease 3, 159–168 (1996) Article No. 0016

REVIEW The Gene Defects Responsible for Familial Alzheimer’s Disease Rudolph E. Tanzi, Dora M. Kovacs, Tae-Wan Kim, Robert D. Moir, Suzanne Y. Guenette, and Wilma Wasco Genetics and Aging Unit, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, 149, 13th Street, Charlestown, Massachusetts 02129 Received September 11, 1996, accepted for publication September 11, 1996

INTRODUCTION Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the central nervous system (CNS) leading to gradual loss of memory, reasoning, orientation, and judgment. The confirmed diagnosis of AD requires the presence of two major neuropathological lesions: neurofibrillary tangles and b-amyloid deposition in brain. A significant portion of AD cases are genetic in etiology (for review see Tanzi et al., 1994; Wasco & Tanzi, 1995) and are termed familial Alzheimer’s disease (FAD). FAD is genetically heterogeneous and in a subset of kindreds is inherited as an autosomal dominant disorder with nearly 100% penetrance. Phenotypically, FAD can be categorized according to age of onset using either 65 years or, more commonly, 60 years for the cutoff between early-onset and lateonset FAD. Genetic analyses have led to the identification of ‘‘causative’’ gene defects in three early-onset (,60 year) FAD genes located on chromosomes 1, 14, and 21, as well as an inherited ‘‘risk factor’’ for late-onset FAD (.60 years) on chromosome 19. b-Amyloid is present in the AD brain in the form of senile plaques and cerebral blood vessel deposits. A wealth of data suggests a central and essential role for the deposition of b-amyloid along with the generation and aggregation of its major component, the Ab peptide (Glenner & Wong, 1984), in the etiology and pathogenesis of AD (for review see Wasco & Tanzi, 1995). The deposition of Ab or specific forms of the peptide appears to be the common pathogenic event which ties together the pathogenetic mechanisms of the various genetic defects leading to AD neuropatho0969-9961/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

genesis. Augmented deposition of b-amyloid is observed in middle-aged patients with Down Syndrome (DS; trisomy 21), most likely owing to the inheritance of three copies of chromosome 21 which contains the gene for the amyloid b-protein precursor (APP; Tanzi et al., 1987). Six different mutations have been identified in the APP gene (Goate et al., 1991; Levy et al., 1990; Murrell et al., 1991; Chartier-Harlin et al., 1991; Hendriks et al., 1992; Mullen et al., 1992), all lying near or within the Ab domain. These mutations have been shown to nearly 100% penetrant and can thus be considered ‘‘deterministic’’ for early-onset FAD. Overall, the APP mutations are responsible for only a small proportion (roughly 2%) of all published cases of FAD (Tanzi et al., 1992) and roughly 5–7% of early-onset FAD. Transgenic mice expressing the APP717V = F mutation have been showed to produce b-amyloid deposits in the form of classical senile plaques accompanied by neuronal and synaptic loss and gliosis (Games et al., 1995) supporting the pathogenicity of the FAD APP mutations. The majority of early-onset FAD cases have been associated with mutations in two novel genes which have been termed presenilin 1 (PS1) on chromosome 14 and presenilin 2 (PS2) on chromosome 1 (Sherrington et al., 1995; Levy-Lehad et al., 1995; Rogaev et al., 1995). Plasma and fibroblasts from patients as well as at-risk carriers harboring mutations in the presenilins have been shown to contain increased amounts of the longer, more amyloidogenic version of the Ab peptide referred to as Ab1–42 or Abx–42 (Scheuner et al., 1996). The final piece of genetic evidence implicating a central and essential role for Ab production, aggregation, and deposition in

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160 AD concerns the probable role of the apolipoprotein E gene, on chromosome 19, in AD neuropathology. Inheritance of the e4 allele (APOE4) of the APOE gene is associated with increased ‘‘risk’’ for late-onset (.60 years) FAD (Saunders et al., 1993; for review, see Wasco & Tanzi, 1995). The impact of APOE4 on AD and age of onset was recently assessed in 310 families with FAD (Blacker et al., 1996). While APOE4 was found to be strongly associated with AD, the risk for AD conferred by APOE4 was most marked in FAD families with mean age of onset between 61 and 65 years. Additionally, while individuals with two copies of APOE4 had a lower age of onset than those with one or no copies (66.4 years vs 72.0 years, P , 0.001), age of onset in individuals with one copy of APOE4 did not differ from those with none. Thus, as a risk factor, APOE4 exerts its maximal effect prior to age 70 and the inheritance of two copies is required to lower the mean age of onset of AD in these families (Blacker et al., 1996). APOE4 is also associated with increased amyloid burden in AD and DS patients who carry an APOE4 allele when compared to those patients who are APOE4-negative (Hyman et al., 1995). Thus, the collective data emanating from studies of the effects of gene defects associated with FAD show that the common denominator in FAD resulting from the APP, PS1, PS2, and APOE genes is the increased deposition of the Ab (or Ab1–42) peptide. Despite these findings, others have argued that the generation of b-amyloid is only a secondary consequence of AD neuropathology (Roses, 1994). Although this is possible, it is also conceivable that processing of APP leading to Ab concurrently results in a deleterious gain of function. A primary role for b-amyloid in AD neuropathogenesis has been strengthened by the finding that aggregates of Ab can be neurotoxic to neurons (Yankner et al., 1990a,b; Frautschy et al., 1991; Cottman et al., 1993; Pike et al., 1993). However, it remains unclear as to whether this is a direct effect of the aggregated peptide or due to the induction of secondary events such as glial/microglial activation, inflammation, free radical generation, and complement activation. It also remains unclear as to exactly which Ab supramolecular structures are capable of inducing neurodegeneration. Some have argued that senile plaques represent ‘‘tombstones’’ and are simply pathogenic markers of AD neuropathology. However, it is important to consider that while it may require many years or decades to form a senile plaque which is visible to the eye of a neuropathologist, undetectable intracellular or extracellCopyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ular aggregates of Ab may induce neuronal cell death many years before the visible plaque appears. In this scenario, undetectable aggregates of Ab would either directly or indirectly induce neurodegeneration, and be subsequently recruited into a senile plaque which could then be regarded as a ‘‘tombstone.’’

THE APP GENE AND Ab Many attempts to define the events underlying the deposition of b-amyloid have been aimed at elucidating the pathways involved in the intracellular trafficking and processing of APP (for review, see Gandy & Greengard, 1994). Following maturation in the endoplasmic reticulum and Golgi (Weidemann et al., 1989), APP is metabolized via one of at least three pathways. In the ‘‘a-secretase’’ pathway, APP is cleaved within the Ab domain preventing formation of b-amyloid and leading to the secretion of the ectodomain of APP (Weidemann et al., 1989; Sisodia et al., 1990; Esch et al., 1990). The remaining C-terminal fragment is then internalized and degraded in the endosomal–lysosomal pathway (Esch et al., 1990; Nordstedt et al., 1993; Golde et al., 1992; Haass et al., 1992a; Caporaso et al., 1992). The ‘‘b-secretase’’ pathway leads to a proteolytic clip at the N-terminus of the Ab domain yielding a C-terminal APP fragment containing intact Ab. Additional cleavage by g-secretase at the C-terminus of the Ab is then necessary for the secretion of Ab (Seubert et al., 1993; Golde et al., 1992; Nordstedt et al., 1991; Gandy et al., 1992; Tamaoka et al., 1992; Knops et al., 1992; Shoji et al., 1992; Haass et al., 1992b). The generation of Ab has been shown to require the internalization of APP (Koo & Squazzo, 1994) most likely via clathrin-coated pits which require the NPXY sequence in the cytoplasmic domain of APP. Deletion of this motif impairs endocytosis of cell-surface APP (Lai et al., 1995). Recently, we employed the yeastbased screening system known as the ‘‘interaction trap’’ to isolate a gene encoding a protein that appears to interact with the NPXY motif of APP (Guenette et al., 1996). The gene, human FE65L (hFE65L) encodes a human homologue of the rat FE65 gene and together with two other human homologues make up a human hFE65/hFE65L gene family. Rat FE65 was initially suggested to be a transcriptional factor, but also contains a WW domain and two phosphotyrosine binding (PTB) domains, motifs normally found in signal transduction proteins. PTB domains bind to the sequence NPXY if the tyrosine is phosphorylated. This suggests hFE65L most likely binds to the NPXY motif in the

Gene Defects in Alzheimer’s Disease

cytoplasmic domain of APP. Thus hFE65L could potentially affect clathrin-coated pit internalization APP and consequently b-amyloid formation. Studies addressing this possibility are under way. The low density lipoprotein receptor-related protein (LRP) has been shown to bind secreted APP containing the KPI domain, leading to its internalization and degradation (Kounnas et al., 1995). Since LRP is believed to be the major APOE receptor in the CNS and delivers lipid to neurons during synaptogenesis following neuronal cell injury (Rebeck et al., 1993), LRP may represent a convergence point for APP and APOE. LRP has been localized to pre- and postsynaptic plasma membranes where it may play a role in preventing synaptic degradation subsequent to neuronal injury clearing glial and neuronal-derived proteases (Kim et al., 1995). It will be important in future studies to determine whether LRP can internalize full-length APP and whether variants of APOE may differentially interfere with this process. Mechanistically, FAD mutations in APP have been shown to alter the secretion of Ab in transfected cells and patient fibroblasts (Cai et al., 1993; Suzuki et al., 1994; Citron et al., 1992). Codon 717 mutations lead to overproduction of Ab1–42, while the ‘‘Swedish’’ FAD mutant of APP leads to an overall increase in Ab secretion. Interestingly, in polarized cells, 80–90% of a-secretase-cleaved APP is released from the basolateral surface (Haass et al., 1994), while APP carrying the Swedish mutation is secreted primarily at the apical surface (Mellman et al., 1995; Selkoe et al., 1995). Thus FAD mutations in APP not only induce changes in the profile of Ab release but may also alter the intracellular trafficking and overall maturation/processing of APP. Elucidation of the factors modulating these pathways will undoubtedly lend valuable clues about the process of b-amyloid deposition. The recent discovery of two novel early-onset FAD genes, the presenilins, may ultimately provide some of the most valuable clues regarding this process.

THE PRESENILIN GENES While PS1 (chromosome 14) was identified by a ‘‘positional cloning’’ strategy (Schellenberg, 1992; Sherrington et al., 1995), PS2 (chromosome 1) was isolated based on its homology to PS1 (Levy-Lehad et al., 1995). Both presenilins are serpentine proteins with six to nine predicted transmembrane domains (Fig. 1; Sherrington et al., 1995; Levy-Lehad et al., 1995; Rogaev et al., 1995). While 67% amino acid identity and the

161 similar structure of the presenilins predict common functions for these proteins, two nonhomologous regions would appear to impart specificity to PS1 and PS2. These two divergent regions are the N-terminus and the large hydrophilic loop between predicted transmembrane (TM) domains six and seven (assuming a seven-TM model) which are located on the same side (cytoplasmic; S. S. Sisodia, personal communication) of the membrane. Northern blot analyses reveal two messages for PS1 at approximately 2.7 and 7.5 kb (Sherrington et al., 1995) and two for PS2 at 2.3 and 2.6 kb (Levy-Lehad et al., 1995b; Rogaev et al., 1995). At the genomic level, PS1 has been shown to comprise 12 exons spanning approximately 75 kb, with the open reading frame limited to exons 3–12 and spanning approximately 24 kb (Clark et al., 1995; Cruts et al., 1996). Alternative splice forms have been identified for PS1 and PS2 in which exon 8 is spliced out have also been reported (Sherrington et al., 1995; Rogaev et al., 1995). An additional PS1 transcript lacking four amino acids (VRSQ) at the 38 end of exon 3 has also been reported (Clark et al., 1995). Alternative splicing of in-frame acceptor sites in introns 9 and 10 of PS2 have also been shown to result in the deletion of Glu324 or replacement of Arg358 with the sequence Ser-Gln-Gly (LevyLahad et al., 1996). Both PS1 and PS2 are ubiquitously expressed. Only the 2.3-kb PS2 message is detected in brain, placenta, lung, and liver, while both transcripts are detected in heart, skeletal muscle, and pancreas (Levy-Lahad et al., 1995; Rogaev et al., 1995). In rat brain, PS1 is expressed predominantly in neurons (Kovacs et al., 1996). Exitotoxic lesions abolish detectable levels of PS1 mRNA which is absent in reactive astrocytes (Page et al., 1996). Highest amounts of PS1 message are observed in the rat hippocampal formation, cerebellar granule cell layers, and choroid plexus followed by adjacent areas of cortex, striatum, and midbrain (Kovacs et al., 1996), while fibrous astrocytes and oligodendrocytes in the white matter do not express detectable levels of PS1 mRNA. PS1 has also been localized to neurons in the mouse brain (Moussaoui et al., 1996). In human brain, the temporal lobe exhibits similar patterns of in situ hybridization for PS1 and PS2, with both expressed in neuronal laminae of the parahippocampal gyrus and the hippocampal formation, and in choroid plexus adjacent to the hippocampal formation (Kovacs et al., 1996). These findings imply that FAD resulting from PS mutations most likely commences in neuronal cell populations. The neuronal staining pattern for PS1 in human brain is similar to that of APP695. PS1 is Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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FIG. 1. A composite of the presenilin proteins based on the seven-TM model. The locations of PS1 mutations are shown as red squares or as the red triangle; the locations of the PS2 mutations including the N1411 Volga German (indicated in box) are shown as purple squares. Note the two clusters of mutations in TM-2 and the N-terminal region of HL-6. Some PS1 squares represent codons (total of six) that contain more than one missense mutation in different FAD kindreds.

expressed more highly in brain areas that are vulnerable to AD neuropathological changes than in areas that are spared in AD (Page et al., 1996). However, the PS1 expression pattern in human is not sufficient to account for selective vulnerability in the AD brain, and PS1 mRNA levels do not differ in AD and control brains (Page et al., 1996).

THE PRESENILIN MUTATIONS Thirty-three different pathogenic mutations in over 60 unrelated pedigrees have now been identified in PS1 and 2 mutations have been detected in two unrelated PS2 families (Table 1; Sherrington et al., 1995; Wasco et al., 1995a; Clark et al., 1995; Cruts et al., 1995, 1996; Campion et al., 1995; Levy-Lehad et al., 1995b; Rogaev et al., 1995; Van Broeckhoven, 1995; Hutton et Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

al., 1996; Boteva et al., 1996; Perez-Tur et al., 1996). All except one of these are missense mutations which result in single amino acid changes. The other mutation destroys a splice acceptor site for exon 9 of PS1 leading to an in-frame deletion of the exon and an amino acid substitution. The mean age of onset in PS1-linked FAD pedigrees is approximately 45 years (range 32 to 56 years), while the average age of onset in the Volga German families with the N141I PS2 mutation is 52 years with individual onset ages ranging from 40 to 85 years. Mutations in PS1 appear to be fully penetrant (with one reported exception; Rossor et al., 1996) and individual PS1 FAD kindreds exhibit a more narrow range of onset age than families possessing the two known FAD mutations in PS2. APOE genotype appears to have no effect on the phenotype of PS1 mutations (unpublished results; Van Broeckhoven et al., 1994). This is in contrast to the situation with APP,

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Gene Defects in Alzheimer’s Disease TABLE 1 Presenilin Mutations Mutation

No. Kindreds

PS1 A79V V82L V96F Y115C Y115H E120K M139I M139V M139T I143F I143T M146L M146V H163Y H163R

1 1 1 1 1 1 1 4 1 1 1 10 3 1 6

I213T A231T A246E L250S A260V C263R P264L P267S R269H E280G E280A

1 1 1 1 1 1 1 1 1 2 5

A285V L286V S289C (exon 9 del) E318G G384A

1 2 1

L392V C410Y PS2 N141I M239V

1 3

Ethnicity Caucasian Caucasian Caucasian Caucasian Caucasian UK Caucasian UK/German Caucasian UK Belgian Italian Italian/UK/Finnish Swedish American/Canadian/Japanese/ Swedish Caucasian Caucasian Nova Scotian UK Japanese Caucasian Caucasian UK Caucasian UK Columbian/Japanese Japanese German/Israeli UK

Domain/ exon TM-1/4 TM-1/4 TM-1/4 HL-1/5 HL-1/5 HL-1/5 TM-2/5 TM-2/5 TM-2/5 TM-2/5 TM-2/5 TM-2/5 TM-2/5 HL-2/6 HL-2/6

TM-4/7 TM-5/7 TM-6/7 TM-6/7 TM-6/7 HL-6/8 HL-6/8 HL-6/8 HL-6/8 HL-6/8 HL-6/8 HL-6/8 HL-6/8 HL-6/8 HL-6/9 HL-6/11

1 2

German Caucasian/Japanese/Belgian Italian Ashkenazi Jewish

7 1

Volga German Italian

TM-2 TM-5

HL-6/11 TM-7/11

Note. Exon numbering is according to that presented in Clark et al. (1995). Protein domains follow the seven-TM model. Abbreviations: TM, transmembrane domain; HL, hydrophilic loop. The ethnicity group Caucasian denotes mixed European ancestry.

where APOE genotype appears to affect the age of onset and degree of amyloid burden in carriers of APP FAD mutations (Sorbi et al., 1993). While FAD mutations are scattered throughout the PS1 gene, all occur in amino acids that are conserved between PS1 and PS2 and are located within, or close

to, TM domains (Fig. 1, Table 1). In fact, roughly half of all reported mutations in PS1 are located in predicted TM domains. Of the mutations reported to date in PS1, 33 involve missense mutations which occur in 26 codons. The 2 mutations in PS2 are in two different codons. Two main clusters of mutations are observed in PS1: one in exon 5 and the other exon 8 (Fig. 1). Exons 5 and 8 contain 10 and 9 mutations, respectively (19 total) or close to 60% of the known PS1 mutations. Small differences in the average age of onset are observed for these mutation clusters vs other mutations in PS1. Seven of the 10 exon 5 mutations are contained in predicted TM2 and carriers have a mean age of onset of 40 years. Twelve of the mutations are in the large hydrophilic loop (HL-6) encoded partly by exons 8–11 and the mean age of onset in carriers is 43 years. Meanwhile, all other mutations in PS1 occur in families with a mean age of onset of 47 years. Thus the mutations in the two hot spots appear to lead to an earlier mean age of onset. Twenty-five of the 35 reported FAD mutations in PS1 and PS2 occur in single kindreds and are thus considered to be genetically ‘‘private’’ mutations. This makes the potential for genetic diagnosis of new cases quite difficult since the data indicate that the probability is over 70% that a new case or family with early-onset FAD is due to a novel PS mutation. Thus, testing for the known presenilin gene mutations as a method for diagnosing new cases of early-onset FAD would most likely not be very useful at this juncture. It also becoming increasingly clear that mutations in the APP and the presenilin genes do not account for all cases of early-onset FAD. We recently sequenced exon 8 in 25 early-onset FAD pedigrees and 95 single cases of early-onset AD (of which 63 had a known family history of the disease) for which the mutations remained unknown. Since roughly 30% of the known PS1 mutations have been found in exon 8, if this trend were to continue and if the majority of the cases sequenced did involve mutations in PS1, one would expect to observe over 30 known or novel mutations in this exon. Surprisingly, we found only one mutation in exon 8, a novel missense mutation (R269H) in a single FAD case with age of onset at 47 years. Based on this finding and the recent experience of other laboratories (Cruts et al., 1996), FAD mutations in PS1 may be less frequent than originally believed. Alternatively, the collection and screening of primarily early-onset FAD cases may be introducing a certain degree of ascertainment bias into the analysis. Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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THE PRESENILIN PROTEINS The PS1 and PS2 genes encode predicted proteins of 463 and 448 amino acids, respectively. We have recently been engaged in an analysis of the proteolytic processing and degradation pathways of the presenilins in stably transfected inducible human H4 neuroglioma cell lines (Kim et al., 1996). Analysis of cell lines expressing PS2 under the control of a tetracyclineresponsive transactivator shows PS2 to be expressed as a 53- to 55-kDa full-length protein. PS2 is proteolytically cleaved into two polypeptides: a 35-kDa Nterminal fragment and a 20-kDa C-terminal fragment (PS2-CTF). The C-terminal fragment is concentrated in the detergent-resistant cellular fraction, consistent with an association with the cytoskeleton or other detergentresistant cellular structures (e.g., caveoli). Degradation of the presenilins can occur via the ubiquitin-proteasome pathway based on our observation that 20S proteasome blockers (ALLN or lactacystin) lead to the accumulation of high molecular weight, polyubiquitinated PS2 in vivo (Kim et al., 1996). Interestingly, blocking the proteasome also leads to accumulation of the 20-kDa C-terminal fragment of PS2 in the detergentresistant cell fraction. Cells expressing PS2 containing the N141I (Volga German) FAD mutation also gave rise to increased amounts of PS2-CTF (compared to cells expressing wild type PS2) in the detergent-resistant fraction. This is apparently due to slower turnover of the C-terminal fragment in the cell line expressing the mutant PS2. Interestingly, the amount of PS2-CTF in the detergent-resistant cellular fractions of wild-type vs mutant PS2-expressing cells could effectively be ‘‘equalized’’ by treating the cells with a 20S proteasome inhibitor. These findings suggest that the Volga German (N141I) PS2 FAD mutation leads to abnormal turnover of PS2 leading to the accumulation of the PS2-CTF in the detergent-resistant cellular fraction. The increased stability of the PS2-CTF specifically in this cell fraction implies that the increased accumulation of this fragment may occur in association with the cytoskeleton or other detergent-resistant cellular structures such as caveoli. The data also support the notion that decreased turnover of the C-terminal fragment in this fraction is due to inefficient degradation of mutant PS2 via the ubiquitin-proteasomal pathway. Like PS2, PS1 is also observed as four different species by Western analysis, a 45- to 50-kDa full-length polypeptide, a high molecular weight aggregate, and two proteolytic cleavage products: a 28-kDa Nterminal fragment and a 19-kDa C-terminal fragment (Thinakaran et al., 1996; Kim et al., 1996). These findCopyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

Tanzi et al.

ings have also been reported in transgenic mice expressing PS1 where proteolytic cleavage was found to be a highly regulated (Thinakaran et al., 1996). It is not yet clear whether FAD mutations in PS1, like the PS2 N141I mutation, lead to abnormal degradation of PS1 and accumulation of PS1 proteolytic fragements. However, as we observed for PS2, the C-terminal fragment of PS1 also accumulates in the detergent-resistant cellular fraction, and PS1 appears to be polyubiquitinated in vivo and degraded by the proteasome pathway. Thus, it will be important to determine in future experiments whether PS1 proteolytic fragments also accumulate at elevated levels as a result of PS1 FAD mutations. Both PS1 and PS2 are localized to the nuclear envelope, the endoplasmic reticulum (ER), and the Golgi in COS and H4 cells (Kovacs et al., 1996). For PS1 and PS2, the staining pattern is reticular and punctate, reminiscent of vesicular staining; no staining of the plasma membrane is observed for either PS1 or PS2. While no gross abnormalities in subcellular localization have been observed for PS1 and PS2 containing FAD mutations, cells expressing PS1 with the A246E FAD mutation revealed diffuse and irregular staining of the nuclear envelope in comparison to the uniform perinuclear staining observed with cells expressing wild-type PS1 (Kovacs et al., 1996). Cook et al. (1996) have shown PS1 to be localized to the rough ER as well as in dendrites but not axons of NT2N human neuronal cell lines infected with human PS1. Additionally, PS1 has been shown not to be glycosylated (Cook et al., 1996).

PRESENILIN FUNCTION The actual biological and physiological roles of the presenilins are not known. Both are serpentine proteins which could conceivably function as receptors, ion channels or pores, or intracellular membrane proteins with specific functions. Some clues about possible function can be drawn from the finding that both presenilins share homology with two proteins of Caenorhabditis elegans: sel-12 (50% identity; Levitan & Greenwald, 1995) and spe-4 (25% identity; L’Hernault & Arduengo, 1992). Greater than 80% of the FAD missense mutations in the presenilin genes occur in residues that are conserved in sel-12. Sel-12 was originally isolated as a facilitator of lin-12, a member of the Notch family of receptors involved with intercellular signaling associated with determining cell fate in the nematode (Levitan & Greenwald, 1995). The SEL-12 protein may act as a coreceptor for LIN-12, aid in the signaling pathway from plasma membrane to nucleus

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during cell differentiation, or play a role in receptor trafficking and recycling (Levitan & Greenwald, 1995). The latter possibility is more consistent with our findings that PS1 and PS2 localize to the ER and Golgi where they might function in protein processing and/or cellular trafficking. Further support for this possibility comes from the finding that loss-of-function mutations in the other nematode presenilin homologue, spe-4, disrupt delivery of proteins to spermatids during spermatogenesis in nematode (L’Hernault & Arduengo, 1992), supporting a role for spe-4 in the cytoplasmic trafficking proteins. The similarity of PS1 and PS2 with these proteins suggests that they may also play roles in the trafficking or recycling of proteins or, alternatively, in the transmission of intercellular signals from the plasma membrane to nucleus. Interestingly, FAD mutations in APP that lead to relative increases in the ratio of Ab42:Ab40 have been shown to also alter intracellular trafficking and/or sorting of APP (Mellman et al., 1995; Selkoe, 1995). Extracellular levels of Abx–42(43) are also increased in the plasma and fibroblasts taken from patients with FAD-associated mutations in PS1 or PS2 (Scheuner et al., 1996). In the brain of a FAD case with the PS1 mutation R269H, a significantly increased degree of amyloid burden is observed. Taken together, these data beg the question of whether the presenilins participate in the processing and/or trafficking of APP and, more specifically, whether FAD mutations in the presenilins steer APP metabolism toward the increased production of Ab or Ab1–42. Our observation of decreased turnover and increased accumulation of the C-terminal PS2 cleavage fragment in the detergentresistant cell fraction of cells expressing PS2-N141I suggests that alterations in the processing or degradation of PS2 predates changes in the abnormal secretion of Ab species. Along these same lines, PS1 containing the FAD mutation resulting in the deletion of exon 9 has been shown to be resistant to normal proteolytic cleavage in lymphoblasts extracted from the blood of an affected carrier (Thinakaran et al., 1996). Further study will be necessary to determine the potential effects of FAD mutations on presenilin processing and degradation and ultimately to define the mechanism by which APP metabolism is altered to give rise to increased levels of Ab1–42.

the pathogenic mutations in the early-onset FAD genes, APP, PS1, and PS2 directly cause AD with nearly 100% penetrance, in a larger subset of AD cases with onset over 60 years (maximally for onset at 61–65 years), inheritance of the APOE4 allele confers increased risk for AD but is not sufficient to cause the disease. Together, these four genes appear to account for approximately 50% of FAD cases. We are actively screening the genome for additional FAD loci by genotyping markers in over 400 FAD nuclear pedigrees and affected sib-pairs (83% late-onset and 17% early-onset). We have recently discovered genetic linkage to a novel FAD locus on chromosome 12 as well as another putative locus on chromosome 3 (unpublished findings). Positional cloning strategies are currently under way to identify these potentially novel FAD genes. A common event which is associated with all of the known FAD genes is the excessive accumulation of the Ab peptide and deposition of b-amyloid in the brain. Thus, a common pathogenic pathway for AD neuropathogenesis appears to center around the cellular trafficking, maturation, and processing of APP, and the subsequent generation, aggregation, and deposition of Ab (or more specifically, Ab1–42). APP and presenilin gene mutations most likely act as either gain-offunction or dominant negative gene defects which may ultimately lead to the transport of APP into intracellular compartments that promote the enhanced production of Ab or Ab1–42. AD patients who carry an APOE4 allele experience increased amyloid burden in their brains compared to APOE4-negative AD cases. Thus, the presence of APOE4 would also appear to lead to abnormal generation, aggregation, or clearance of Ab in the brain Ab, perhaps by working in concert with its neuronal receptor, LRP. While the exact mechanisms by which the known FAD gene changes lead to the onset of AD remain unclear, the available data indicate that novel therapies aimed at curbing the generation, aggregation, and deposition of Ab would appear to carry the greatest potential for the effective treatment of this formidable disease.

ACKNOWLEDGMENTS SUMMARY Four different genes have now been found to contain AD-associated mutations or polymorphisms. While

This work was supported by grants from the NIA, NINDS, the American Health Assistance Foundation, and the Metropolitan Life Foundation. R. Tanzi is a Pew Scholar, and T.-W. Kim and D. M. Kovacs are recipients of National Research Service Awards.

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