Molecular and cellular biology of Alzheimer amyloid

J Mol Neurosci (1992) 3:111-125 Journal of Molecular Neuroscience �9 Birkh~iuser Boston 1992

Molecular and Cellular Biology of Alzheimer Amyloid

Char les A. M a r o t t a , Rona ld E. Ma jocha , and B a r b a r a T a t e Department of Psychiatry and Program in Neuroscience, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114, USA

Abstract. Alzheimer's Disease (AD), a disorder of unknown etiology, is the most common form of adult-onset dementia and is characterized by severe intellectual deterioration. The definitive diagnosis of AD is made by postmortem examination of the brain, which reveals large quantities of neurofibrillary tangles (NFT) and senile plaques within the parenchyma. The NFT are composed of paired helical filaments associated with several cytoskeletal proteins. The primary protein component of senile plaques is 13/A4 amyloid, a 42-43 amino acid peptide derived from a much larger molecule, the amyloid precursor protein (APP). Vascular 13/A4 amyloidosis is also prevalent in the disease. The mechanism by which [3/A4 amyloid accumulates in the AD brain is unknown. Recent research has demonstrated that the precursor molecule, APP, is a transmembrane protein with a large extracytoplasmic domain, a membrane spanning region that includes the portion that gives rise to 13/A4 amyloid, and a short intracytoplasmic domain. The precursor has multiple forms among which are those that differ by a variable length insert within the extracytoplasmic domain. The insert has sequence homology to the family of Kunitz protease inhibitor proteins. Cellular and animal models have been developed to study the nature of APP processing and the biological and'behavioral consequences of 13/A4 amyloidosis. The results of such studies indicate that the normal processing of APP involves enzymatic cleavage of the molecule within the 13/A4 amyloid region, thus pre- venting the accumulation of 13/A4 in the normal brain. The factors leading to abnormal processing of APP, and con- sequent 13/A4 amyloid accumulation within the AD brain, have yet to be identified. In cell culture, the biological effects associated with 13/A4 amyloid include neurotrophic and neurotoxic activities, while the peptide has also been shown to have dramatic behavioral effects in animal models.

Alzheimer's Disease (AD) is a syndrome characterized by intellectual deterioration occurring in an adult that is severe enough to interfere with occu-

Offprint requests to: C.A. Marotta

pational or social performance (Katzman, 1986). It is the most common form of adult-onset dementia and is the fourth leading cause of death in the United States (Hay and Ernst, 1987). Commonly observed cognitive changes in AD include not only impairments in memory, but also disturbances in language use, perception, the ability to learn necessary skills, solve problems, think abstractly, and make judgmen t s (Ka t zman , 1986). Pe r sona l i ty changes, e.g., paranoia, may occur in some patients. The earliest symptom has been described as an impairment in the ability to retain information over a brief delay (Lishman, 1978; Wells, 1977)o Neuropsychological examinat ions have revealed that information decays at a faster rate in AD patients than in patients with other types of memory disorders (Hart et al., 1988; Moss et al., 1986). These observations have been correlated with the neuropathological changes that are found in the brain. The major input and output pathways of the hippocampus have strikingly high concentrat ions of the lesions of AD, viz., senile plaques (SPs) and neurofibrillary tangles (NFTs) , suggesting that the functional isolation of the hippocampus may be responsible for the memory impairment (Hyman et al., 1984). There is also a substantial loss of neurons (Mountjoy et al., 1983) and a decrease in acetylcho- line positive sites (Coyle et al., 1983; Davies and Matoney, 1980; Perry, 1980). However , the lesions of AD are not confined to a single brain region or neurotransmitter system. Norepinephrine, seroto- nin, somatostatin, and glutamate also have been implicated in this disorder (Davies et al., 1980; Katz- man, 1986; Price, 1986; Sasaki et al., 1986), as have neurotrophic factors (e.g., ne rve growth factor, NGF). Clinical and neuropharmacological studies have been reviewed elsewhere (Greenwald et al., 1983; Katzman, 1986; Price, 1986). A recent review has summarized the data on the role of N G F in AD (Perry, 1990).

112 Marotta et al.: Alzheimer Amyloid

The cause or causes of AD are not as yet identified. Both genetic and environmental factors, and their interaction, are implicated. Head trauma, infectious agents, and neurotoxins (e.g., aluminum) may play a role in the development of disease with the characteristics of AD (Mortimer et al., 1985; Perl and Brody, 1980; Wisniewski et al., 1981). Met- abolic abnormalities also have been described (Vinters, 1987). For example, using fluorine-18(2- fluro-2-deoxy-D-glucose, FDG), diminished glucose utilization was observed in temporal and parietal cortex in Alzheimer's disease (Friedland et al., 1983; Koss et al., 1985).

Although there are currently accepted clinical guidelines for the probable diagnosis of AD (Mc- Khann et al., 1984), the definitive diagnosis is made by postmortem examination of the brain, which reveals the presence of abundant numbers of NFTs and SPs. NFTs are composed of partly insoluble protein polymers described as paired helical filaments (PHFs) (Kidd, 1963; Wisniewski et al., 1976) that occur within affected neurons. The PHFs accumulate in the perikarya of large neurons as 10-nm protein fibers that are helically twisted around one another with a crossover at 80 nm and that are ar- ranged in a double-helical stack (Kidd, 1963; Kosik, 1991; Marotta, 1984; Wischik et al., 1985). Although tau, neurofilament, and ubiquitin epitopes appear to be associated with PHFs (Anderton et al., 1987; Kosik, 1991; Lee et al., 1990; Love et al., 1988; Nikina, 1989; Wischik et al., 1988), it also has been suggested that the filaments are derived from amyloid fibrils (Masters et al., 1985a). More extensive descriptions of NFTs and their importance to the pathophysiology of AD are reported elsewhere (Crowther and Wischik 1985; Kosik, 1991; Landon and Kidd, 1989).

The remainder of this review will focus on amytoid found in the AD brain.

Senile Plaques

Amyloid-containing senile plaques are a prominent feature of selective areas of the AD brain (Divry, 1927; Wisniewski and Terry, 1973) and the Down's syndrome brain. They range in size from approxi- mately 9 o.m to 50 Cm in diameter, when viewed by immunocytochemical methods designed to detect amyloid, and they vary in morphology and density (Majocha et al., 1988). Classical staining methods demonstrate SPs as large as 200 Ixm (Tomlinson and Corsellis, 1984). They are most often found in the cerebral cortex, but they also occur in deeper grey

matter, including the amygdaloid nucleus, the cor- pus striatum, and the diencephalon. Plaques also have been described in the cerebellum (Pro et al., 1980; Rudelli and Wisniewski, 1985). SPs are composed of extracellular amyloid, reactive cells, and degenerating neurites that contain PHFs, lyso- somes, abnormal mitochondria, and astrocytic processes (Wisniewski and Terry, 1973). The mechanisms responsible for the excessive accumulation of amyloid, the major proteinaceous component of SPs, have begun to be addressed at the levels of protein chemistry, molecular biology, and genetics, as discussed subsequently.

Amyloid is composed of fibrils of 4-8 nm diameter that form the core of SPs (Merz et al., 1983). The amyloid is readily demonstrated by application of thioflavin S or Congo red to brain sections. In the latter case, polarized light causes amyloid to appear with a characteristic yellow-green color. The staining property reflects the presence of twisted beta pleated sheet fibrils. A detailed discussion of the biochemistry and histochemistry of amyloid has been presented by Glenner (1980).

Vascular Amyloidosis

Vascular amyloidosis, referred to as congophilic angiopathy, has been recognized since the early part of this century as a significant aspect of the microscopic pathology of Alzheimer's disease (Tomlin- son and Corsellis, 1984; Vinters, 1987). Over 90% of Alzheimer cases have congophilic angiopathy (Glenner et al., 1981; Ishi et al., 1983; Lee and Stemmerman, 1978). Similar to parenchymal amyloid deposits, vascular amyloid is demonstrated by characteristic thioflavin S and Congo red staining reactions. The parieto-occipital cortex is usually more affected than that in the frontal and temporal lobes (Tomlinson and Corsellis, 1984).

The amyloid appears to infiltrate the microvas- culature; affected vessels often pass from the lep- tomeninges into the cortex. Small cerebral vessels with arterioles that appear as thickened tubes are observed. The changes include the small pial and intracortical arterioles, the leptomeningeal vessels, and the intracortical capillaries (Tomlinson and Corsellis, 1984). Immunocytochemical and electron microscopic studies have indicated that the amyloid component of senile plaques is often observed in close proximity to affected microvessels (Allsop et al., 1986; Majocha et al., 1988). However, the an- g iopathy may occur wi thou t senile p laques (Mandybur, 1975; Mountjoy et al., 1982).

Marotta et al.: Alzheimer Amyloid 113

[3/A4-Amyloid Peptide

Glenner and Wong (1984a) partially identified the structure of amyloid fibrillary protein derived from meningeal vessels of an AD brain; a 4.2-kilodalton (kDa) peptide, known as 13-amyloid, was isolated and shown to have a unique sequence of 28 amino acids. A peptide of nearly identical structure was subsequently isolated from the cerebrovasculature of a Down's syndrome brain (Glenner and Wong, 1984b); a single amino acid substitution, of glutamic acid for glutamine at position 11, distinguished the two peptides. Similar results were obtained inde- pendently by Masters et al. (1985b), who partly purified and analyzed amyloid plaque cores from the AD cerebral cortex; a peptide with an amino acid sequence nearly identical to ~3-amyloid was referred to as A4 amyloid. The isolated 4.2-kDa peptide was observed to be relatively insoluble in a variety of solvents (Masters et al., 1985b); the solubility was later shown to be pH dependent (Barrow and Za- gorski, 1991). Short synthetic peptides with structures homologous to [3-amyloid or A4-amyloid are interesting from a physical--chemical perspective since they exhibit aggregation properties that re- semble the naturally occurring amyloid of 4.2 kDa Castano et al., 1986; Gorevic et al., 1987; Kirschner et al., 1986; Salim et al., 1988).

The terms 13-amyloid and A4 are often used in- terchangeably. For the purposes of the present discussion, the parent molecule from which the depos- ited form of amyloid is derived will be referred to as the amyloid precursor protein (APP). The segment that is excised and contributes to senile plaques, composed of 42 or 43 amino acids (Kang et al., 1987), will be designated 13/A4.

may account for the nonuniform observations from different laboratories using different reagents. The use of monoclonal antibodies (Mabs) to 13/A4 com- bined with computer enhancement methods applied to affected brain tissues facilitates studies on molecular mechanisms involved in amyloid accumulation and provide improved neuropathological aids for the identification of potential AD subtypes (Ma- jocha et al., 1988).

As discussed in detail in a subsequent section, the precursor of [3/A4, the APP, is a transmembrane protein occurring in various forms, including a protein of 695 amino acids. Polyclonal antibodies (Pabs) raised against various APP sites were applied, along with anti-t3/A4 Mabs, in conjunction with selective cytochemical staining methods, to control and AD cases (Tate-Ostroff et al., 1989a, 1990). It was noted that a subset of SPs contained both 13/A4 and additional APP antigens (including the N-terminal region, a segment within the extracytoplasmic domain, and a C-terminal site). The re- suits support the view that APP segments other than 13/A4 undergo processing and subsequent deposition in SPs. We can speculate that processing of the entire molecule occurs at an initial stage of amyloid accumulation; however, the resistance of the [3/A4 domain to proteolysis after it is abnormally cleaved, unlike the remainder of the molecule, al- lows this peptide to persist and accumulate. Pro- posed mechanisms for amyloid deposition during plaque formation may benefit from taking into account processing of the entire APP molecule, in addition to the 13/A4 region, rather than be confined exclusively to consideration of the transmembrane site.

Distribution of [3/A4 Antigen

Using polyclonal antiserum to a synthetic [3/A4 peptide containing residues 1-10, it was shown that SP amyloid shares antigenic determinants with the similar fibrillary lesion of cerebral vessels (Glenner and Wong, 1984b). The same antiserum failed to detect neurofibriUary tangles. By contrast, results from a second laboratory indicated that antiserum raised against residues 1-11 of the peptide failed to detect vascular amyloid or SPs but, rather, exhibited exclusive specificity for the NFT, and antiserum to the 13/A4 peptide extending from residues 11 to 23 stained both SPs and blood vessels (Masters et al., 1985a). Thus, various antibodies to [3/A4 are non- identical with regard to immunospecificity, and this

Expression of Amyloid Precursor Protein mRNA

Cloned probes for the APP gene are commonly available. Using in situ hybridization methods, several reports have focused on amyloid mRNA in control and AD brains with attention to RNA levels, differential expression in various cell types, and relationship to pathologic markers characteristic of AD (Chou et al., 1990; Cohen et al., 1988; Goedert, 1987; Higgins et al., 1988; Lewis et al., 1988; Palm- ert et al., 1988). It is generally observed that cortical and subcortical neurons in normal brains have substantial levels of APP mRNA. In certain instances, in situ hybridization data led to the conclusion that the amyloid mRNA was decreased in AD relative to controls when frontal cortex was examined, and this result was consistent with Northern blot anal-


yses (Bahmanyar et al., 1987; Goedert, 1987). How- ever, other studies have led to the opposite conclusion concerning m R N A concentrations in cortical and/or subcortical areas (Cohen et al., 1988; Hig- gins et al., 1988; Lewis et al., 1988; Palmert et al., 1988). Certain neurons of the AD brain may contain relatively abundant amyloid mRNA at some point during the course of the disease. High levels of amyloid mRNA appear to be synthesized by AD cortical neurons that are morphologically intact, and these cells are not necessarily coincident with the cortical laminar pattern that is typical of amyloid deposits observed with anti-[3/A4 Mabs (Chou et al., 1990). Further, there is no obvious relationship between neurons containing abundant amyloid mRNA and the distribution of plaques identified by thioflavin S staining. While the neuronal synthesis of amyloid may be a significant factor at some point during plaque formation, it may not be the exclusive determinant. This conclusion is consistent with data derived from amyloidotic transgenic mice (Wirak et al., 1991).

These findings raise the possibility that transport of amyloid away from cells of origin to sites of deposition may be a meaningful aspect of the molecular pathology of AD. This interpretation provides a foundation for exploring a relationship between intracellular amyloid overproduction and distant sites of deposition. Immunocytochemical investigations led to the suggestion that diffusional processes, possibly secondary to secretion, may be involved in extracellular amyloid accumulation (Benes et al., 1989; Tate-Ostroff et al., 1989a). This view is supported by the identification of secreted forms of APP (Rumble et al., 1989).

Amyloid Precursor Protein Gene

The APP gene is present in a single copy per haploid genome and is located on the long arm of chromosome 21 (Goldgaber et al., 1987; Kang et al., 1987; Robakis et al., 1987; Tanzi et al., 1987a). As discussed subsequently, the gene for the familial form of AD also has been located on this chromosome (Tanzi et al., 1987a,b). The APP gene is greater than 50 kb and consists of 18 exons and 17 introns (Le- maire et al., 1989). Multiple forms of APP arise by alternative splicing of primary transcripts of a single precursor gene (Kitaguchi et al., 1988; Kang and Muller-Hill, 1990). APP is expressed in mammals in both neuronal and nonneuronal cells and tissues (Goedert 1987; Shivers et al., 1988; Tate-Ostroff et al., 1989a; Zimmerman et al., 1988). Rat and mouse APP have 97% homology to human APP, and high

concentrations of rat APP are found in areas of the brain commonly affected by AD (Card et al., 1988; Shivers et al., 1988; Yamada et al., 1987). The APP is ubiquitous across species. Recently the Droso- phila homolog of APP was described (Luo et al., 1990).

It is now known that multiple forms of the APP exist; they are referred to as APP-695, APP-751, and APP-770; numbers indicate the amino acid residues (Kitaguchi et al., 1988; Ponte et al., 1988; Tanzi et al., 1988). In the extracytoplasmic region, there is a variable segment of 56 residues inserted at residue 289, which corresponds to APPs larger in size than 695 residues (Kitaguchi et al., 1988; Ponte et al., 1988; Tanzi et al., 1988). This site shows identity with the Kunitz family of protease inhibitors (KPI). Kitaguchi et al. (1988) described the APP-770 variant, which contains a sequence of 19 amino acid residues contiguous with the KPI region. Subsequently APP-563, derived from an AD brain, was reported that contains the KPI domain in which 208 amino acids at the C-terminus are re- placed by 20 amino acids derived from nucleotide sequences with homology to the Alu repeat family (de Sauvage and Octave, 1989). While the significance of this variant is not known, it was suggested to represent a secreted form of the 13/A4 peptide that lacks the transmembrane domain. An additional APP of 714 amino acids also has been reported (Gode et al., 1990).

Amyloid Precursor Protein Gene Product

13/A4 amyloid that accumulates in the AD brain is a peptide that is derived from the APP, first identified by molecular cloning of the APP-695 DNA (Goldgaber et al., 1987; Kang et al., 1987; Robakis et al., 1987; Tanzi et al., 1987a; Vitek et al., 1988; Zain et al., 1988). From the cloning data a model for the APP was constructed by Kang et al. (1987) in which the APP has a large extracytoplasmic domain, a transmembrane domain (which gives rise to the [3/A4 deposition product of 4.2 kDa) and an intracytoplasmic domain. The signal sequence, for transport through the endoplasmic reticulum membrane, is followed by a region rich in cysteine, which suggests that disulfide bridges may stabilize this portion of the structure. Within the next 100 residues is a stretch of seven uninterrupted threo- nine residues and a region containing 28 glutamic acid residues and 17 aspartic residues. This domain could bind cations extensively and may have physiological significance. Sodium dodecyl sulfate (SDS) may be bound to a lesser extent than usual


due to this domain. This possibility would account for the unexpected electrophoretic mobilities of APPs analyzed by SDS-polyacrylamide gel electro- phoresis (PAGE) (see below). The region from residue 290 to 597, at which point the 13/A4 site begins, contains two potential N-glycosylation sites at po- sitions 467-469 and 496-498. The 13/A4 peptide (residues 597-638 or 639) is either 42 or 43 amino acids in length and partly includes the putative transmembrane domain (amino acids 625--648). The C-terminal region of the APP is relatively small, consisting of 57 residues, when compared with the length of the extracytoplasmic domain of nearly 600 amino acids.

Following the transmembrane region, lysine residues are present (residues 649-651) that could in- teract with phospholipid head groups in the membrane (Kang et al., 1987). This feature has been described for the junction between membrane and cytoplasmic domains of cell-surface receptors. One site (amino acids 684--686) is a potential glycosylation sequence. HeLa cells transfected with APP-695 cDNA contain proteins with masses of 91,103, 110, and 130 kDa. The protein of 110 kDa appeared to corresponded to an N- and O-glycosylated form of the APP-695 (Weidemann et al., 1989). In vitro expression of the APP-695 in cell-free systems in the presence of microsomal membranes generates an N-glycosylated protein of 91 kDa (Dyrks et al., 1988). Exper imenta l ly determined molecular weights for APP are higher than the theoretical val- ues of 78.6 and 88.6 kDa, respectively, for different APP forms (Dyrks et al., 1988; Kang et al., 1987; Kitaguchi et al., 1988) (described below). As men- tioned, this is presumably due to the potential cation binding properties of the abundant aspartic and glutamic acid residues.

During in vitro stiidies of synthetic peptides corresponding to the cytoplasmic domain, it was observed that protein kinase C rapidly catalyzed the phosphorylation of a peptide corresponding to amino acid residues 645-661 on Ser-655 (Buxbaum et al., 1990). Thus, this site may be an important control region for amyloid metabolism and its interaction with other intracellular regulatory elements.

Amyloid Precursor Protein and Genetics of AD

Compelling evidence in support of a familial form of AD comes from restriction fragment length poly- morphism (RFLP) analyses that indicate linkage between DNA markers on chromosome 21 and auto- somal dominant familial Alzheimer disease (FAD)

(St. George-Hyslop et al., 1990a). However, nega- tive LOD scores on chromosome 21 for subsets of pedigrees as well as linkage with chromosome 19 loci were also reported (Schellenberg et al., 1988). Thus, at present, FAD appears to be genetically heterogeneous; a number of families segregate a gene defect on chromosome 21 while others may involve other loci or a nongenetic etiology.

Although the APP gene was mapped to chromosome 21 (Goldgaber et al., 1987; Kang et al., 1987; Robakis et al., 1987) near an FAD locus (St. George-Hyslop et al., 1990a), detailed analysis revealed that the FAD and APP loci are not tightly linked (Tanzi et al., 1987b). Nevertheless, subsequent reports emphasized the possibility that amyloid is the etiologic agent in certain forms of familial disease.

An unusual amyloid precursor structure may contribute to the high content of amyloid plaques in the AD brain. Cloning studies utilizing cortical poly(A) + RNA extracted from the brain of a sporadic case of AD showed that APP mRNA matched the coding and noncoding regions of APP mRNA from non-AD sources (Vitek et al., 1988; Zain et al., 1988). In the sporadic case the structure of the KPI site was also identical to the non-AD sequence (Chou et al., 1988; Vitek et al., 1988). Subsequently the structure of APP from certain familial cases came under scrutiny. Linkage of the APP gene was examined in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), an au- tosomal dominant form of cerebral amyloid angiopathy characterized by amyloid deposition in iepto- meningeal arteries and cortical arterioles, which leads to early death of afflicted individuals. RFLP analyses indicated that the APP gene is tightly linked to HCHWA-D (Van Broeckhoven et al., 1990). Concurrently, cloning and sequencing of two amyloid exons from patients with this form of amyloidosis revealed a single amino acid substitution (Glu instead of Gln) at [3/A4 position 22, which occurs outside the transmembrane domain (Levy et al., 1990). Subsequently, an APP exon from an early-onset FAD case was sequenced and shown to contain a missense mutation in which Ile is substi- tuted for Val within the transmembrane domain, separated by two residues from the C-terminus of the 13/A4 peptide (Goate et al., 1991). While two FAD cases contained the mutation, 100 unrelated individuals and 14 cases of familial late-onset disease failed to demonstrate the substitution. Based upon these data, it can be hypothesized that muta- tions in the APP gene can predispose to AD, but are not the only condition that influences the possibility of developing the disease.


Cellular Models of Amyioidosis and Processing of Amyioid Precursor Protein

Studies on the normal processing of APP have indicated that proteolytic cleavage is by a pathway that would not normally produce the intact ~/A4 fragment found in SPs. COS-I cells were transfected with APP homologs of various lengths and the minimal sequence for APP cleavage was identified (Sisodia et al., 1990). The data indicated that normal processing releases fragments smaller than 13/A4. A different approach (Esch et al., 1990) led to a similar overall conclusion. Human embryonic 293 cells were transfected with APP-695 and 751 and N- and C-terminal fragments of soluble and membrane- bound forms were purified and sequenced. It was observed that constitutive processing occurred within the 13/A4 sequence. Similar to the aforemen- tioned conclusion, this proteolytic cleavage could not account for the production of intact [3/A4 in the AD brain. These results appear relevant to the ob- servation that APPs are found in serum and cerebrospinal fluid, where they appear to occur as normal secretion products that are shorter than the full- length molecules (Palmert, 1989a; Rumble et al., 1989).

Recently, synthetic substrates were used to identify cathepsin B as the normal processing enzyme, APP secretase (Tagawa et al., 1991). These data, as well as numerous speculations, have stimulated at- tempts to identify the putative aberrant proteolytic mechanism that is responsible for production of the amyloidogenic [3/A4 peptide in the AD brain. This unfolding field of research is reviewed elsewhere (Ishiura, 1991).

The PC12 cell line, and other cultured cells, ex- press APP on the cell surface and secrete APP peptides, smaller than t h e p a r e n t molecule, among which is protease nexin-II (Autillio-Gambetti et al., 1988; Marotta et al., 1989a,b; Oltersdorf et al., 1989; Palmert et al., 1989a,b; Refolo et al., 1989; Tate-Ostroff et al., 1989a,b; Van Nostrand et al., 1989; Weidemann et al., 1989). Thus this cell type has been used extensively as a model system for gaining insight into the processing of amyloid that may be of potential significance for understanding aspects of the molecular pathogenesis of AD. PC 12 cells display neuronal properties, including neurite extension, when exposed to nerve growth factor (NGF). When stimulated with NGF for one week, the cells cease to multiply and begin extending processes (Greene and Tischler, 1976). Investigations into the relationship between NGF and amyloid production have led to inconsistent results. Palmert

et al. (1988), using developing brain, and Mobley et al. (1988), using PCI2 cells, reported an increase in amyloid mRNA upon NGF addition. However, Wion et al. (1988) dispute this claim since, in their study, NGF did not increase the cellular content of the APP transcript in PC12 cells.

Refolo et al. (1989) confirmed the usefulness of utilizing NGF to probe amyloid metabolism. Similar to other results (Marotta et al., 1989b), these authors found that PC12 cells release APP as proteins of 140 and 105 kDa. Prolonged treatment with NGF led to bands of 120-125 kDa. Other workers have found that in the absence of NGF, PC12 cells normally shed APP bands of 115 kDa (Majocha et al., 1991; Weidemann et al., 1989). A current view is that NGF is a modulator of APP production and/or processing. With regard to a potential regulatory role of NGF, it appears significant that protease nexin-II is able to form complexes with the gamma subunit of NGF (Bothwell and Shooter, 1978; Knauer et al., 1982, 1983; Oltersdorf et al., 1989).

Experiments on transfected cells appear particu- larly useful since a major obstacle to clarifying molecular mechanisms involved in amyloid metabolism in AD has been the unavailability of practical laboratory models for this uniquely human disorder. Thus, to examine the cellular basis of amyloid accumulation, a number of genetically engineered cell lines that overexpress APP domains have been established (Marotta et al., 1989a,b). Immunoelec- tron microscopy of transfected PC12 cells demonstrated that the f3/A4 epitope was inserted into the cell membrane of transfectants (Tate-Ostroff et al., 1989b).

13/A4: Trophic or Toxic?

Studies on the effects of conditioned media from transfected PCI2 cells that overexpress amyloid have shown that they stimulate neurite outgrowth (Zain et al., 1989). While the effective factor has not yet been identified, the data are not inconsistent with the results of Allsop et al. (1988), who suggested that the APP is processed to release a peptide ligand such as a neuroendocrine hormone or growth factor. However, other data are less uni- form with regard to the stimulatory effects of APP subdomains and the consequences of transfection on secretory products.

For example, Whitson et al. (1989) demonstrated that a peptide homologous to the first 28 amino acids of 13/A4, 13/A4 (1-28), exhibited neurotrophic activity towards hippocampal pyramidal neurons since it enhanced neuronal survival under the cul-


ture conditions used. Subsequently it was shown that 13/A4 (1-42), when applied at high concentrations (100 ixg/ml) to El8 rat hippocampal cultures, increased neurite length and branching (Whitson et al., 1990). This result appears to contrast directly with the report of Yankner et al. (1989), who transfected PC12 cells with portions of the APP DNA. Conditioned media from transfectants was toxic to neurons in primary hippocampal cultures, and the toxic agent was immunologically identified as the amyloid peptide. The report of a toxic effect appears to conflict with an earlier report (Yankner et al., 1988) using the same experimental system. The toxic effect is also inconsistent with the data of other workers (Saitoh et al., 1989), who observed that release of amyloid protein from transfected cells was stimulatory to growth. Schubert et al. (1989) reported that the secreted form of the APP that contains the protease inhibitor sequence is a mitogen for 3T3 cells, while the form of APP lacking the Kunitz site is not.

In an attempt to reconcile trophic vs. toxic activity of amyloid peptides, both functional roles were examined in the same experimental system (Yankner et al., 1990). The viability of rat El8 hippocampal cells was measured in response to vary- ing doses of 13/A4 (1--40) and length of time in culture. When added within one day of plating, cellular survival increased for a period of two days; in older cultures (greater than three days) the peptide activity was toxic. It was concluded that [3/A4 is neurotrophic only during the early stages of differentia- tion. However, there was a strong dose-dependent effect: 13/A4 (1-40) at 0.1 nM was not toxic irrespective of when it was added to the cultures; the toxic response was observed at 40 nM. Amino acids 25-35 were identified as the trophic and/or toxic fragment while C-tei:minal amino acids were inac- tive, in contrast to a previous report (Yankner et al., 1989). The effects of [3/A4 were selectively re- versed by specific tachykinin neuropeptides. It was suggested that 13/A4 is a tachykinin antagonist op- erating via the substance P receptor.

The relationship between NGF and amyloid was explored further using cultured chick and rat sympathetic and sensory neurons. The biological effects of amyloid plaque core protein (APCP) consisting mainly of 13/A4 as well as purified 13/A4 were examined in culture. APCP and 13/A4 were toxic to neurons whose survival was dependent upon NGF but toxicity was reversible at low doses; sprouting was also prevented in freshly plated neurons (Roher et al., 1991). These results, together with other observations that administration of NGF to animals and

cells is known to increase levels of 13/A4, led the authors to suggest that the clinical use of NGF as a treatment modality for AD patients may be more harmful than efficacious.

While it is not as yet conclusive that the 13/A4 peptide itself is neurotoxic in the human brain, there is evidence that destruction of cellular elements occurs in the vicinity of SPs as indicated by double-stain immunocytochemistry performed on both early and late Alzheimer tissues. In general, there were fewer axons within plaques and around SPs than in adjacent areas and their normal morphology was disrupted. (Benes et al., 1991). These data are consistent with the view that amyloidotic SPs or a factor (factors) associated with them may be injurious to neurons.

Amyloid and Animals Models

In certain aged rodents that exhibit defined behavioral deficits, cholinergic neurons of basal forebrain nuclei were examined since atrophy of these cells is a common finding in AD (Coyle et al., 1983). In aged rats that exhibit spatial memory deficits (but not those without behavioral impairments) the APP mRNAs containing the KPI motif (APP-751, APP- 770) are increased relative to the noninserted form (APP-695), as judged by in situ hybridization (Hig- gins et al., 1990). The data suggest that overexpression of KPI-containing APP is related to neuronal atrophy and subsequent behavioral disturbances.

A direct approach to relating APP to behavioral deficits is to administer homologous peptides to animals that are then tested in a behavioral paradigm. Mice were given either intracerebroventricular or intrahippocampal injections of [3/A4 (1-28) immedi- ately after foot shock avoidance training (Flood et al., 1991). In a dose-dependent manner the mean number of trails necessary to achieve performance was greater for peptide-injected mice than for mice receiving vehicle alone. Shorter fragments of 13/A4 produced the same effect. The data were interpreted as decreased retention of long-term memory due to administration of 13/A4 peptides rather than interference with short-term memory as in the case of AD patients.

Cell adhesion processes are implicated in memory formation, including long-term memory, at the level of synaptic remodeling, which can be studied by administration of antiserum to membrane glyco- conjugates. Using this approach, anti-APP antiserum (prepared against a 14-amino acid peptide of an extracytoplasmic site) was infused intraventricu-


larly into rats that were trained to avoid stepping down to an electroshock by remaining on a platform for a specified period (Doyle et al., 1990). De- creased step-down latency occurred at 24- and 48- hour recall times when antiserum was administered 2.5 hours following training, but not at 4 or 6 hours. The data were interpreted as interference with task acquisition, rather than memory consolidation or retrieval. Antiserum to neurofilament or to the [3/A4 peptide had no effect on recall. The results suggest that APP, but not [3/A4, is associated with the early phase of memory formation.

In the previous section cellular models of amyloidosis were discussed with respect to the production of trophic or toxic agents that most likely include 13/A4 amyloid. The genetically transformed cell model was recently adapted to a behavior paradigm (Tate et al., 1991). Since AD patients suffer circadian rhythm dysfunction, this behavioral defi- cit was modeled in rat by a cell-grafting technique. PC 12 cells transfected with the f3/A4-C-terminal region of the APP were implanted into the suprachi- asmatic nuclei (SCN) of rats; the SCN is a primary circadian oscillator in mammals. Animals receiving amyloidotic cell grafts, but not animals receiving control cell grafts, exhibited disrupted activity rhythms; temperature rhythms were unaffected. The specificity of the disruption was similar to circadian dysfunction seen in AD patients. The data supported an association between a defined behavioral disruption and amyloid overexpression either directly or through the release of cellular factors as a consequence of amyloid overproduction.

Among the most promising approaches to eluci- dating the contribution of amyloid to neuropathology is the preparation of transgenic mice that carry an overabundance of the APP gene. Several approaches are possible. These include insertion of DNA with homologous vs. heterologous promoters for amyloid, insertion of DNA of APP variants with or without the KPI, or use of subdomains of the APP gene that include the ~3/A4 region. Two recent reports demonstrate accumulation and deposition of amyloid in the mouse brain irrespective of the promoter and the APP segment selected.

The rat neural-specific enolase promoter was linked to the full-length APP-751, containing the protease inhibitor domain, for the preparation of transgenic mice (Quon et al., 1991). 13/A4 immunoreactive material was significantly increased in brain neurons and neuropil (as fine puncta) relative to controls as well as in neuronal processes of hippocampus; full-length APP was also present in neuritic processes. Immunoreactive deposits were evi-

dent in cortex and hippocampus as both compact and diffuse (amorphous) deposits. This work suggested that regulation of APP-751 levels may be one mechanism to generate amyloid deposits. A second report indicated that 13/A4 DNA fused to a fragment from the 5' region of the human APP gene, which directs neuron-specific expression, is sufficient to cause fibril formation (Wirak et al., 1991). As a result human [3/A4 was synthesized and accumulated in the CNS of mice at six months of age. 13/A4 immunoreactivity was predominantly in the hippocampus (CA1 and CA2), rather than the cortex, and was also observed in some blood vessels. EM revealed fibrillary material within cells and processes that resembled those in the AD brain. The data indicate that other factors, in addition to [3/A4 accumulation, are necessary for the extracellular accumulation of AD-like deposits of amyloid, and this process may be developmentally regulated. Both reports on the production of transgenic mice indicate that amyloid fibril formation in brain occurs after overexpression of the amyloid gene.

Biological Mechanisms and Amyloidosis

The normal function of APP is unknown at this time. However, a series of recent experiments indicated that the larger APPs appear to be protease nexin-II (Oltersdorf et al., 1989; Van Nostrand et al., 1989), a protease inhibitor that stimulates neurite outgrowth (Oltersdorf et al., 1989; Van Nos- trand et al., 1989). Studies carried out from a different perspective accumulated evidence that APP may have a physiological role in cell-substrate adhesion. Highly purified APP, when coated into Petri dishes, was found to enhance adhesion of PC12 cells (Schubert et al., 1989). As cell adhesion posi- tively influences neurite outgrowth, APP may stimulate this process. It has been speculated that APP functions in cell contact (Shivers et al., 1988) or axonogenesis (Luo et al., 1990). These and other examples of possible roles that have been ascribed to APP (Ishiura, 1991) suggest that the molecule is most likely multifunctional.

Several factors could contribute to amyloid accumulation in the AD brain. These include the rate of transcription of the amyloid gene, the rate of translation of the mRNA, posttranslational processing, intracellular transport, and turnover. Contrib- utory mechanisms most clearly would involve abnormal proteolysis. Each process potentially affects the rate of production of 13/A4 and its deposition into an insoluble form. With respect to the site of


origin and transport, a blood-brain barrier defect has been suggested to occur in AD (Wisniewski et al., 1982), although this has been disputed (McGeer et al., 1987; Rozemuller et al., 1988; Wong et al., 1985). Since anti-amyloid antibodies detect blood vessels as well as plaques (Allsop et al., 1986; Ma- jocha et al., 1988; Wong et al., 1985), it can be ar- gued that this protein has its origin in the serum and gains entry to the brain through damaged vessels, possibly capillaries. Circulating APP, shorter than the full-length molecule (presumably due to normal proteolysis) is found in serum and cerebrospinal fluid (Palmert et al., 1989a). Alternatively, a neuronal origin for amyloid has been proposed: after synthesis, the fibrous protein that is produced is be- l i e v e d to f o r m , o r at l e a s t c o n t r i b u t e to , intraneuronal NFTs (Guiroy et al., 1987; Masters et al., 1985a). It was suggested that as neurites degen- erate, a plaque is formed that eventually invades the capillary wall leading to the characteristic angiopathy. More recently, a major component of NFT, A68, was identified as an abnormally phosphory- lated form of tau protein, a normal cytoskeletal constituent involved in the microtubule network (Lee et al., 1990). In light of the new data, the potential contribution of amyloid to NFTs requires further detailed evaluation.

With regard to a role for amyloid in cellular survival, we can speculate that overproduct ion (due to a transcription and/or translation defect) or overac- cumulation of amyloid (due to a proteolytic processing defect) initiates a cascade of events that af- fec t the viabi l i ty o f the hos t cell. The initial response may be stimulatory to the cell (e.g., neurite extension, etc.) followed by subsequent cellular deterioration and release of 13/A4 fragments and other factors with trophic and/or toxic activity. Se- nile plaque formation and localized areas of cell death may ensue. A local increase in NGF results in fu r the r c o n s e q u e n c e s for amylo id p r o d u c t i o n and/or processing. However , the hypothesized in- teractions may be subject to regulation by KPI- containing APPs since protease nexin-II binds the gamma subunit of NGF; the binding may modulate the formation of certain cleavage products and ag- gregated forms of 13/A4-derived peptides. The ratio of KPI to non-KPI inserts may be a further contrib- utory regulatory factor.

Other hypotheses lead to equally plausible sce- narios concerning the effects of amyloid on cellular viability. By use of genetically engineered cells and animals, we may be able to develop a comprehen- sive and dynamic view of the biological activity of amyloid that takes into account the regulation of

APP levels, the biological activity of individual subdomains, their interaction with other cellular regu- la tory mechan i sms , the m o d u la to ry ef fec ts of trophic factors, the relation of APP processing to neurodegeneration, and the precise role played by this protein in the molecular pathogenesis, and/or etiology of AD.

Acknowledgments. The authors thank Alison Morris for her contribution to the literature evaluation. This work was prepared while C.A.M. was the recipient of grants from the following sources: NIH P01 AG02126, the Ax- elrod Family Fund, and the Metropolitan Life Founda- tion. R.E.M. and B.T. were recipients of Milton Fund Awards from Harvard University.

References

Allsop, D., Landon, M., Kidd, M., Lowe, J.S., Reyn- olds, G.P., Gardner, A. (1986). Monoclonal antibodies raised against a subsequence of senile plaque core protein react with plaque cores, plaque periphery, and cerebrovascular amyloid in Alzheimer's disease. Neu- rosci. Lett. 68:252-256

Allsop, D., Wong, C.W., Ikeda, S.I., Landon, M., Kidd, M., Glenner, G.G. (1988). Immunohistochemical evidence for the derivation of a peptide ligand from the amyloid 13-protein precursor of Alzheimer's disease. Proc. Natl. Acad. Sci. U.S.A. 85:2790-2794

Anderton, B.H., Brion, J.P., Flament-Durand, J., Haugh, M.C., Kahn, J., Miller C.L., Probst, A., Ul- rich, J. (1987). Neurofibrillary tangles and the neuronal cytoskeleton. J. Neural Transm. Suppl. 24:191-196

Autillio-Gambetti, L., Morandi, A., Tabaton, M., Schaetzle, B., Kovaks, D., Perry, G., Greenberg, B., Gambetti, P. (1988). The amyloid precursor protein of Alzheimer disease is expressed as a 130 kDa polypep- tide in various cultured cell types. FEBS Lett. 241:94-98

Bahmanyar S., Higgins, G., Goldgaber, D., Lewis, D.A., Morrison, J.H., Wilson, M.C., Shankar, S.K., Gaj- dusek, D.C. (1987). Localization of amyloid 13 protein messenger RNA in brains from patients with Alzhei- mer's disease. Science 237:77-80

Barrow, C.J., Zagorski, M.G. (1991). Solution structures of 13 peptide and its constituent fragments: Relation to amyloid deposition. Science 253:17%182

Benes, F.M., Reifel, J.L., Majocha, R.E., Marotta, C.A. (1989). Evidence for diffusion of Alzheimer amyloid A4 (13-amyloid) epitope during neuritic plaque formation. Neuroscience 33:483--488

Benes, F.M., Farol, P.A., Majocha, R.E., Marotta, C.A. (1991). Evidence for destruction of axons within regions occupied by senile plaques in Alzheimer cortex. Neuroscience 42:651-660


Bothwell. M.L., Shooter, E.M. (1978). Thermodynamics of interaction of the subunits of 7 S nerve growth factor. The mechanism of activation of the esteropeptid- ase activity by chelators. J. Biol. Chem. 253:8458- 8464

Buxbaum, J.D., Gandy, S.E., Cicchetti, P., Ehrlich, M.E., Czernik, A.J., Fracasso, R.P., Ramabhadran, T.V., Unterbeck, A.J., Greengard, P. (1990). Pro- cessing of Alzheimer beta/A4 amyloid precursor protein: Modulation by agents that regulate protein phosphorylation. Proc. Natl. Acad. Sci. USA 87:6003- 6006

Card, J.P., Meade, R.P., Davis, L.G. (1988). Immunocy- tochemical localization of the precursor protein for 13-amyloid in the rat central nervous system. Neuron 9:835-846

Castano, E.M., Ghiso, J., Prelli, F., Gorevic, P.D., Migheli, A., Frangione, B. (1986). In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to Alzheimer's disease 13-protein. Biochem. Biophys. Res. Commun. 141: 782-789

Chou, W.G., Majocha, R.E., Sajdel-Sulkowska, E.M., Benes, F.M., Salim, M., Stoler, M., Fulwiler, C.E., Ventosa-Michetman, M., Rodenrys, A.M., Moore, K.E., Webb, T., Zain, S.B., Marotta, C.A. (1988). Immunologic and molecular genetic studies on amyloid deposition in the Alzheimer's disease brain. Brain Dysfunct. 1:133-145

Chou, W.-G., Zhu, W., Salim, M., Rehman, S., Tate- Ostroff, B., Majocha, R.E., Sajdel-Sulkowska, E.M., Marotta, C.A., Zain, S.B. (1989). Extracytoplasmic and A4 domains of the amyloid precursor protein: Mo- lecular cloning, genetically engineered cell lines and immunocytochemical investigations. Alzheimer's Dis- ease and Related Disorders . K. Iqbal, H.M. Wisniewski, B. Winblad (eds). Alan R. Liss, New York, pp 991-999

Chou, W.G., Zain, S.B., Rehman, S., Tate-Ostroff, B., Majocha, R.E., Benes,.F.M., Marotta, C.A. (1990). Alzheimer cortical neurons containing abundant amyloid mRNA. Relationship to amyloid deposition and senile plaques. J. Psychiatr. Res. 24:37-50

Cohen, M.L., Golde, T.E., Usiak, M.F., Younkin, L.H., Younkin, S.G. (1988). In situ hybridization of nucleus basalis neurons shows increased 13-amyloid mRNA in Alzheimer's disease. Proc. Natl. Acad. Sci. U.S.A. 85:1227-1231

Coyle, J.T., Price, D.L., DeLong, M.R. (1983). Alzhei- mer's Disease: A disorder of cortical cholinergic in- nervation. Science 219:1184-1190

Crowther, R.A., Wischik, C.M. (1985). Image recon- struction of the Alzheimer paired helical filament. EMBO J 4:3661-3665

Davies, P., Maloney, A.J.F. (1980). Selective loss of central cholinergic neurons in Alzheimer's disease. Lan- cet 2:1403

Davies, P., Katzman, R., Terry, R.D. (1980). Reduced somatostatin-like immunoreactivity in cerebral cortex

from cases of Alzheimer's disease. Nature 288:279-- 280

de Sauvage, F., Octave, J.N. (1989). A novel mRNA of the A4 amyloid precursor gene coding for a possibly secreted protein. Science 245:651-653

Divry, P. (1927). Etude histo-chimique des plaques senile. J. Neurol. Psych. 27:643-657

Doyle, E., Bruce, M.T., Breen, K.C., Smith, D.C., Anderton, B., Regan, C.M. (1990). Intraventricular in- fusions of antibodies to amyloid-13-protein precursor impair the acquisition of a passive avoidance response in the rat. Neurosci. Lett. 115:97-102

Dyrks, J.D., Weidemann, A., Multhaup, G., Salbaum, J.M., Lamaire, H.G., Kang, J., Muller-Hill, B., Mas- ters, C.L., Beyreuther, K. (1988). Identification transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer's Disease. EMBO J. 7:949-- 957

Esch, F.S., Keim, P.S., Beattie, E.C., Blacher, R.W., Cltlwell, A.R., Oltersdorf, T., McClure, D., Ward, P.J. (1990). Cleavage of amyloid 13 peptide during constitutive processing of its precursor. Science 248:1122- 1124

Flood, J.F., Morley, J.E., Roberts, E. (1991). Amnestic effects in mice of four synthetic peptides homologous to amytoid 13 protein from patients with Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 88:3363-3366

Friedland, R.P., Budinger, T.F., Ganz, E., Yano, Y., Mathis, C.A., Koss, B., Ober, B.A., Huesman, R., Derenzo, S. (1983). Regional cerebral metabolic alterations in dementia of the Alzheimer-type: Positron emission tomography with 18-fluorodeoxyglucose. J. Comput. Assist. Tomogr. 7:590-589

Glenner, G.G. (1980). Amyloid deposits and amyloidosis. The 13-fibrilloses. N. Engl. J. Med. 302:1333-1343

Glenner, G.G., Wong, C.W. (1984a). Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 120:885-890

Glenner, G.G., Wong, C.W. (1984b). Alzheimer's disease and Down's syndrome: Sharing of a unique cerebrovascular amyloid fibril protein. Biochem. Biophys. Res. Commun. 122:1131-1135

Glenner, G.G., Henry, J.H., Fujihara, S. (1981). Congo- philic angiopathy in the pathogenesis of Alzheimer's degeneration. Ann. Pathol. 1:120-129

Goate, A., Chartier-Harlin, M.-C., Mullan M.L., Brown, J., Crawford, F., Fidani, L., Giuffra, L., Haynes, A., Irving, N., James, L., Mant, R., Newton, P., Rooke, K., Roques, P., Talbot, C., Pericak-Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M., Hardy, J. (1991). Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature 349:704--706

Gode, T.E., Estus, S., Usiak, M., Younkin, L.H. , Younkin, S.G. (1990). Expression of 13 amyloid protein precursor mRNA: Recognition of a novel alternatively spliced form and quantitation in Alzheimer's disease using PCR. Neuron 4:253-267


Goedert, M. (1987). Neuronal localization of amyloid beta protein precursor mRNA in normal human brain and in Alzheimer's disease. EMBO J. 6:3627-3632

Goldgaber, D., Lerman, M.I., McBride, O.W., Saffiotti, U., Gajdusek, D.C. (1987). Characterization and chro- mosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease. Science 235:877-880

Gorevic, P.D., Castano, E.M., Sarma, S., Frangione, B. (1987). Ten to fourteen residue peptides of Alzhei- mer's disease protein are sufficient for amyloid fibril formation and its characteristic x-ray diffraction pattern. Biochem. Biophys. Res. Commun. 147:854-862

Greene, L.A., Tischler, A.S. (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromo- cytoma ceils which respond to nerve growth factor. Proc. Natl. Acad. Sci. U.S.A. 73:2424-2428

Greenwald, B.S., Mohs, R.C., Davis, K.L. (1983). Neu- rotransmitter deficits in Alzheimer's Disease: Criteria for significance. J. Am. Geriatr. Soc. 31:310--316

Guiroy, D.C., Miyazaki, M., Multhaup, G., Fischer, P., Garruto, R.M., Beyreuther , K., Masters, C.L., Simms, G., Gibbs, C.J., Gajdusek, D.C. (1987). Amy- loid of neurofibrillary tangles of Guamanian parkin- sonism-dementia and Alzheimer disease share identical amino acid sequence. Proc. Natl. Acad. Sci. U.S.A. 84:2073-2977

Hart, R.P., Kwentus, J.A., Harkins, S.W., Taylor, J.R. (1988). Rate of forgetting in mild Alzheimer's type dementia. Brain Cogn. 7:31-38

Hay, J.W., Ernst, R.L. (1987). The economic costs of Alzheimer's disease. Am. J. Pub. Health 77:116%1175

Higgins, G.A., Lewis, D.A., Bahmanyar, S., Goldgaber, D., Gajdusek, D.C., Young, W.G., Morrison, J.H., Wilson, M.C. (1988). Differential regulation of amyloid-~3-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 85:1297-1301

Higgins, G.A., Oyler, G.A., Neve, R.L., Chen, K.S., Gage, F.H. (1990). Altered levels of amyloid protein precursor transcripts in the basal forebrain of behav- iorally impaired aged rats. Proc. Natl. Acad. Sci. U.S.A. 87:3032-3036

Hyman, B.T., VanHoesen , G.W., Damasio, A.R., Barnes, C.L. (1984). Alzheimer's disease: Cell specificity pathology isolates the hippocampat formation. Science 225:1168--1170

Ishii, N., Nishihara, Y., Horie, A. (1983). Lobar cerebral hemorrhage and amyloid angiopathy: A report of 4 autopsy cases. No To Shinkei Brain and Nerve 35:167-174

Ishiura, S. (1991). Proteolytic cleavage of the Alzheimer's disease amyloid A4 precursor protein. J. Neurochem. 56:363-369

Kang, J., Muller-Hill, B. (1990). Differential splicing of Alzheimer's disease amyloid A4 precursor RNA in rat tissues: preA4 695 mRNA is predominantly produced in rat and human brain. Biochem. Biophys. Res. Com- mun. 166:1192-1200

Kang, J., Lemaire, J .G., Unterbeck, A., Salbaum, J.M.,

Masters, C.L., Grzeschik, K.H., Multhaup, G., Bey- reuther, K., Muller-Hill, B. (1987). The precursor of Alzheimer's disease amytoid A4 protein resembles a cell-surface receptor. Nature 325:733--736

Katzman, R. (1986). Alzheimer's Disease. N. Engl. J. Med. 314:964-973

Katzman, R., Saitoh, R. (1991). Advances in Alzheimer's disease. FASEB J. 5:278-286

Kidd, M. (1963). Paired helical filaments in electron microscopy in Alzheimer disease. Nature 197:192-193

Kirschner, D.A., Abraham, C., Selkoe, D.J. (1986). X-ray diffraction from intraneuronal paired helical filaments and extraneuronal amyloid fibers in Alzheimer disease indicates cross-beta conformation. Proc. Natl. Acad. Sci. U.S.A. 83:503-507

Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S., Ito, H. (1988). Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity. Nature 331:531-534

Knauer, D.J. , Scaparro, K.M., Cunningham, D.D. (1982). The gamma subunit of 7S nerve growth factor binds to cells via complexes formed with two-cell- secreted nexins. J. Biol. Chem. 257:15098-15104

Knauer, D.J., Thompson, J.A., Cunningham, D.D. (1983). Protease nexins: Cell-secreted proteins that mediate the binding, internalization, and degradation of regulatory serine proteases. J. Cell Physiol. 117:385-396

Kosik, K.S. (1991). Alzheimer plaques and tangles: advances on both fronts. Trends Neurosci. 14:218--219

Koss, E., Friedland, R.P., Ober, B.A., Jagust, W.J. (1985). Differences in lateral hemispheric asymmetries of glucose utilization between early and late-onset Alzheimer-type dementia. Am. J. Psychiatry 142:638- 640

Landon, M., Kidd, M. (1989). Amyloid in Alzheimer's disease. Biochem. Soc. Trans. 17:69-72

Lee, S., Stemmerman, G.N. (1978). Congophilic angiopathy and cerebral hemorrhage. Arch. Pathol. Lab Med. 102:317-328

Lee, V., Balin, B.J., Otvos, L., Jr., Trojanowski, J.Q. (1990). A68: A major subunit of paired helical filaments and derivatized forms of normal tau. Science 251:675-678

Lemaire, H.G., Salbaum, J.M., Multhaup, G., Kang, J., Bayney, R.M., Unterbeck, A., Beyreuther, K., Mul- ler-Hill, B. (1989). The preA4 (695) precursor protein of Alzheimer's disease A4 amyloid is encoded by 16 exons. Nucleic Acids Res. 17:517-522

Levy, E., Carman, M.D., Fernandez-Madrid, I.J., Power, M.D., Lieberburg, I., van Duinen, S.G., Ge- rard, T., Bots, A.M., Luyendijk. W., Frangione, B. (1990). Mutation of the Alzheimer's disease amyloid gene in hereditary cerebral hemorrhage, Dutch type. Science 248:1124-1128

Lewis, D.A., Higgins, G.A., Young, W.G., Goldgaber, D., Gajdusek, D.C., Wilson, M.C., Morrison, J.H. (1988). Distribution of precursor amytoid-beta-protein messenger RNA in human cerebral cortex: Relation-


ship to neurofibrillary tangles and neuritic plaques. Proc. Natl. Acad. Sci. U.S.A. 85:1691-1695

Lishman, A. (1978). Organic Psychiatry. Blackwell, Lon- don

Love, S., Saitoh, T., Quijada, S., Cole, G.M., Terry, R.D. (1988). Alz 50, ubiquitin and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies. J. Neuropathol. Exp. Neurol. 47:393-405

Luo, L., Martin-Morris, L.E., White, K. (1990). Identi- fication, secretion, and neural expression of APPL, a Drosophila protein similar to human amyloid protein precursor. J. Neurosci. 10:3849-3861

Majocha, R.E., Benes, F.M., Reifel, J.L., Rodenrys, A.M., Marotta, C.A. (1988). Laminar-specific distribution and infrastructural detail of amyloid in the Alzheimer cortex visualized by computer-enhanced imaging of unique epitopes. Proc. Natl. Acad. Sci. U.S.A. 85:6182-6186

Majocha, R.E., Tate, B., Ventosa-Michelman, M., Marotta, C.A. (1991). Characterization of neurite- stimulating activity from conditioned media of transfected PC12 cells that overexpress 13/A4-amyloid. Soc. Neurosci. 17:1105

Mandybur, T.I. (1975). The incidence of cerebral amyloid angiopathy in Alzheimer's disease. Neurology 25:20- 126

Marotta, C.A. (1984). Neuronal intermediate filaments. Handbook of Neurochemistry, Vol 7. A. Lajtha (ed). Plenum Press, New York, pp 305-309

Marotta, C.A., Majocha, R.E., Coughlin, J.E., Manz, H.J., Davies, P., Ventosa-Michelman, M., Chou, W.G., Zain, S.B., Sajdel-Sulkowska, E.M. (1986). Transcriptional and translational regulatory mechanisms during normal aging of the mammalian brain and in Alzheimer's disease. Prog. Brain Res. 70:303-320

Marotta, C.A., Chou, W.-G., Majocha, R.E., Watkins, R., Zain, S.B. (1989a). Overexpression of amyloid precursor protein A4 (13-amyloid) immunoreactivity in ge- neticaUy transformed cells. Implications for a cellular model of Alzheimer amyloidosis. Proc. Natl. Acad. Sci. U.S.A. 86:337-341

Marotta, C.A., Walcott, E.C., Tate-Ostroff, B., Ma- jocha, R.E. (1989b). Immunocytochemical studies of brain and PCI2 cells using antibodies to the amyloid precursor protein. Soc. Neurosci. 15:1375

Masters, C.L., Multhaup, G., Simms, G., Pottgiesser, J., Martins, R.M., Beyreuther, K. (1985a). Neuronal origin of a cerebral amyloid: Neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels. EMBO J. 4:2757-2763

Masters, C.L., Simms, G., Weinman, N.A., Multhaup, G., McDonald, B.L., Beyreuther, I. (1985b). Amyloid plaque core protein in Alzheimer's disease and Down syndrome. Proc. Natl. Acad. Sci. U.S.A. 82:4245- 4249

McGeer, E.G., Singh, E.A. , McGeer, P.L. (1987). Gamma-glutamyltransferase: Normal cortical levels in Alzheimer disease. Alzheimer Dis. Assoc. Disorders 1:38--42

McKhann, G., Drachman, D., Folstein, M., Katzman, R., Price, D., Stadlan, E.M. (1984). Clinical diagnosis of Alzheimer's disease: Report of the NINCDS- ADRDA Work Group under the auspices of the DHHS Task Force on Alzheimer 's disease. Neurology 34:93%944

Merz, P.A., Wisniewski, H.M., Somerville, R.A., Bobin, S.A., Masters, C.L., Iqbal, K. (1983). Ultrastructural morphology of amyloid fibrils from neuritic and amyloid plaques. Acta Neuropathol. 60:113-124

Mobley, W.C., Neve, R.L., Prusiner, S.B., McKinley, M.P. (1988). Nerve growth factor increases mRNA levels for the prion protein and the 13-amyloid protein precursor in developing hamster brain. Proc. Natl. Acad. Sci. U.S.A. 85:9811-9815

Mortimer, J.A., French, L.R., Hutton, J.T., Schuman, L.M. (1985). Head injury as a risk factor for Alzhei- mer's disease. Neurology 35:264--267

Mountjoy, C.Q., Tomlinson, B.E., Gibson, P.H. (1982). Amyloid and senile plaques and cerebral blood vessels. A semi-quantitative investigation of a possible relationship. J. Neurol. Sci. 57:8%102

Mountjoy, C.Q., Roth, M., Evans, N.J.R., Evans, H.M. (1983). Cortical neuronal counts in normal elderly con- trois and demented patients. Neurobiol. Aging 4:1-11

Moss, A., Albert, M., Butters, N., Payne, M. (1986). Differential patterns of memory loss among patients with Alzheimer's disease, Huntington's disease and alcoholic Korsakoff ' s syndrome. Arch. Neurol. 43:23%246

Nikina, N. (1989). The reinterpretation of the immuno- chemical study of Alzheimer neurofibrillary tangles. Ann. Med. 21:117-119

Oltersdorf, T., Fritz, L.C., Schenk, D.B., Lieberburg, I., Johnson-Wood, K.L., Beattie, E.C., ward, P.J., Blacher, R.W., Dovey, H.F., Sinha, S. (1989). The secreted form of Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II. Na- ture 341:144-147

Palmert, M.R., Golde, T.E., Cohen, M.L., Kovacs, D.M., Tanzi, R.E., Gusetla, J.G., Usiak, M.F., Younkin, L.H., Younkin, S.G. (1988). Amyloid protein precursor messenger RNAs: Differential expression in Alzheimer's disease. Science 241:1080-1084

Palmert, M.R., Podlisny, M.B., Witker, D.S., Oltersdorf, T., Younkin, L.H., Selkoe, D.J., Younkin, S.G. (1989a). The 13-amyloid protein precursor of Alzheimer disease has soluble derivatives found in human brain and cerebrospinal fluid. Proc. Natl. Acad. Sci. U.S.A. 86:6338--6342

Palmert, M.R., Siedlak, S.L., Berman-Podisny, M., Greenbert, B., Shelton, E.R., Chan, H.W., Usiak, M., Selkoe, D.J., Perry, G., Younkin, S.G. (1989b). Solu- ble derivatives of the ~3 amyloid protein precursor of Alzheimer's disease are labeled by antisera to the amyloid protein. Biochem. Biophys. Res. Commun. 165:182-188

Perl, D.P., Brody, A.R. (1980). Alzheimer's disease: X-ray spectrometric evidence of aluminum accumula-


tion in neurofibrillary tangle-beating neurons. Science 208:297-299

Perry, E.K. (1980). The cholinergic system in old age and Alzheimer's disease. Age Ageing 9:1-8

Perry, E.K. (1990). Nerve growth factor and the basal forebrain cholinergic system: A link in the etiopathol- ogy of neurodegenerative dementias? Alzheimer Dis. Assoc. Disorders 4:1-13

Ponte, P., Gonzalez-DeWhitt, P., Schilling, J., Miller, J., Hsu, D., Greenberg, B., Davis, K., Wallace, W., Lie- berburg, I., Fuller, F., Cordell, B. (1988). A new A4 amyloid mRNA contains a domain homologous to serine protease inhibitors. Nature 331:525-527

Price, D.L. (1986). New perspectives on Alzheimer's disease. Annu. Rev. Neurosci. 9:48%512

Pro, J.D., Smith, C.H., Sumi, S.M. (1980). Presenile Alzheimer disease: Amyloid plaques in the cerebellum. Neurology 30:820-825

RanaUi, P., Bergeron, C. (1984). Amyloid angiopathy in Alzheimer's disease. Ann. Neurol. 16:11%127

Quota, D., Wang, Y., Catalano, R., Scardina, J.M., Mu- rakami, K., Cordell, B. (1991). Formation of [3-amyloid protein deposits in brains of transgenic mice. Na- ture 352:23%241

Refolo, L.M., Salton, S.R., Anderson, J.P., Mehta, P., Robakis, N.K. (1989). Nerve and epidermal growth factors induce the release of the Alzheimer amyloid precursor from PC 12 cell cultures. Biochem. Biophys. Res. Commun. 164:664-670

Robakis, N.K. , Ramakrishna, N., Wolfe, G., Wis- niewski, H.M. (1987). Molecular cloning and characterization of a cDNA encoding the cerebrovascular and neuritic plaque amyloid peptides. Proc. Natl. Acad. Sci. U.S.A. 84:4190-4194

Roher, A.E., Ball, M.J., Bhave, S.V., Wakade, A.R. (1991). 13-Amyloid from Alzheimer disease brains in- hibits sprouting and survival of sympathetic neurons. Biochem. Biophys. Res. Commun. 174:572-579

Rozenmuller, J.M., Eikelenboom, P., Kamphorst, W., Stam, F.C. (1988). L.ack of evidence for dysfunction of the blood-brain barrier in Alzheimer's disease: An immunohistochemical study~ Neurobiol. Aging 9:383- 391

Rumble, B., Retallack, R., Hilbich, C., Simms, G., Mul- thaup, G., Martins, R., Hockey, A., Montgomery, P., Beyreuther, K., Masters, C.L. (1989). Amyloid A4 protein and its precursor in Down's syndrome and Alzheimer's disease. N. Engl. J. Med. 320:1446-1452

Rudelli, R.D., Wisniewski, H.M. (1985). Cerebellar amyloid plaques in Alzheimer's dementia and senile dementia of the Alzheimer type: Morphology and distribution. J. Neuropathol. Exp. Neurol. 44:364

Saitoh, T., Sundsmo, M., Roch, J.-M., Kimura, N., Cole, G., Schubert, D., Oltersdorf, T., Schenk, D.B. (1989). Secreted form of amyloid 13 protein precursor is involved in growth regulation of fibroblasts. Cell 58:615- 622

Salim, M., Zain, S.B., Chou, W-G., Sajdel-Sulkowska, E.M., Majocha, R.E., Rehman, S., Benes, F.M.,

Marotta, C.A. (1988). Molecular cloning of amyloid cDNA from Alzheimer brain messenger RNA. Correl- ative neuroimmunologic and in situ hybridization studies. Familial Alzheimer's Disease Molecular Genetics, Clinical Prospects and Societal Issues. J.P. Blass, G.D. Miner, L.A. Miner, R.W. Richter and J.L. Val- entine (eds). Marcel Dekker, New York, pp 153-165

Sasaki, H., Muramoto, O., Kanazawa, I., Arai, H., Ko- saka, K., Iizuka, R. (1986). Regional distribution of amino acid transmitters in postmortem brains and presenile and senile dementia of Alzheimer type. Ann. Neurol. 19:263-269

Schellenberg, G.D., Bird, T.D., Wiseman, E.M., Moore, D.K., Boehnke, M., Bryant, E.M., Lampe, T.H., Nochlin, D., Sumi, S.M., Deeb, S.S., Beyreuther, K., Martin, G.M. (1988). Absence of linkage of chromosome 21q21 markers to familial Alzheimer's disease. Science 241:1507-1510

Schubert, D., Cole, G., Saitoh, T., Oltersdorf, T. (1989). Amyloid beta protein precursor is a mitogen. Bio- chem. Biophys. Res. Commun. 162:83-88

Shivers, B., Hilbich, C., Multhaup, G., Salbaum, J.M., Beyreuther, K., Seeburg, P.H. (1988). Alzheimer's disease amyloidogenic glycoprotein: Expression pattern in rat brain suggests a role in cell contact. EMBO J. 7:1365-1370

Sisodia, S.S., Koo, E.H., Beyreuther, K., Unterbeck, A., Price, D.L. (1990). Evidence that 13-amyloid protein in Alzheimer's disease is not derived by normal processing. Science 248:492-495

St. George-Hyslop, P.H., Tanzi, R.E., Polinsky, R.J., Haines, J.L., Nee, L., Watkins, P.C. (1990a). The genetic defect causing familial Alzheimer's disease maps on chromosome 21. Science 235:885-890

St. George-Hyslop, P.H., Haines, J.L., Farter, L.A., Po- linsky, R., Van Broeckhoven, C., Goate, A., McLach- lan, D.R., Orr, H., Bruni, A.C., Sorbi, S., Rainero, I., Foncin, J.-F., Pollen, D., Cantu, J.-M., Tupler, R., Voskresenskaya, N., Mayeux, R., Growdon, J., Fried, V.A., Myers, R.H., Nee, L., Backhovens, H., Martin, J.-J., Rossor, J., Owen, M.J., Mullan, M., Percy, M.E., Karlnsky, J., Rich, S., Heston, L., Mon- tesi, M., Mortilla, M., Nacmeas, M., Gusella, J.F., Hardy, J.A., members of the FAD Collaborative Study Group (1990b). Genetic linkage studies suggest that Alzheimer's disease is not a single homogeneous disorder. Nature 347:194-197

Tagawa, K., Kunishita, K., Maruyarna, K., Kazuaki, Y., Kominami, E., Tsuchiya, T., Suzuki, K., Tabira, T., Sugita, H., Ishiura, S. (1991). Alzheimer's disease amyloid 13-clipping enzyme (APP secretase): Identifi- cation, purification, and characterization of the enzyme. Biochem. Biophys. Res. Commun. 177:377-387

Tanzi, R.E., Gusella, J .F. , Watkins, P.C., Gruns, G.A.P., St. George-Hyslop, P., VanKeuren, M.L., Patterson, D., Pagan, S., Kurnit, D.M., Neve, R.L. (1987a). Amyloid [3 protein gene: cDNA, mRNA distribution and genetic linkage near the Alzheimer locus. Science 235:880--884


Tanzi, R.E., St. George-Hyslop, P.H., Haines, J.L., Po- linsky, R.J., Nee, L., Foncin, J.-F., Neve, R.L., Mc- Clatchey, A.I . , Conneally, P.M., Gusella, J.F. (1987b). The genetic defect in familial Alzheimer's disease is not tightly linked to the amyloid [3-protein gene. Nature 329:156-157

Tanzi, R.E., McClatchey, A.I., Lamperti, E.D., Villa- Komaroff, L., Gusella, J.F., Neve, R,L. (1988). Pro- tease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease. Nature 331:528-530

Tate, B., Aboody, K.S., Morris, A.M., Majocha, R.E., Marotta, C.A. (1991). Alterations in the circadian rhythms of rats receiving SCN implants of PC12 cells overexpressing Alzheimer's disease 13-amyloid. Soc. Neurosci. 17:1066

Tate-Ostroff, B., Majocha, R.E., Marotta, C.A. (1989a). Identification of cellular and extracellular sites of amyloid precursor protein extracytoplasmic domain in normal and Alzheimer brains. Proc. Natl. Acad. Sci. U.S.A. 86:745-749

Tate-Ostroff, B., Walcott, E.C., Paskevich, P., Zain, S.B., Chou, W.G., Majocha, R.E., Marotta, C.A. (1989b). Morphological and immunological studies of PC12 ceils transfected with the 13-amyloid region of the amyloid precursor protein. Soc. Neurosci. 15:654

Tate-Ostroff, B., Majocha, R.E., Walcott, E.C., Ven- tosa-Michelman, M., Marotta, C.A. (1990). Colocal- ization of amino terminal and A4 (13-amyloid) antigens in Alzheimer plaques: Evidence for coordinated processing of the amyloid precursor protein. J. Geriatr. Psychiatry Neurol. 3:13%145

Tomlinson, B.E., Corsellis, J.A.N. (1984). Ageing and the dementias. Greenfield's Neuropathology, ed 4. J.H. Adams, H.A.N. Corsellis, L.W. Duchen (eds). John Wiley & Sons, New York, pp 951-1006

Van Broeckhoven, C., Haan, J., Bakker, E., Hardy, J.A., Van Hul, W., Wehnert, A., Vegter-Van der Vlis, M., Roos, R.A.C. (1990). Amyloid 13 protein precursor gene and hereditary cerebral hemorrhage with amyloidosis (Dutch). Science 248:1120-1122

Van Nostrand, W.E., Wagner, S.L., Suzuki, M., Choi, B.H., Farrow, J.S., Geddes, J.W., Cotman, C.W., Cunningham, D.D. (1989). Protease nexin-II, a potent anti-chymotrypsin, shows identity to amyloid 13-protein precursor. Nature 341:546-549

Vinters, H.V. (1987). Cerebral amyloid angiopathy: A critical review. Stroke 18:311-324

Vitek, M.P., Rasool, C.G., de Sauvage, F., Vitek, S.M., Bartus, R.T., Beer, B., Ashton, R.A., Macq. A.-F., Maloteaux, J.-M., Blume, A.J., and Octave, J.-N. (1988). Absence of mutation in the beta-amyloid cDNAs cloned from the brains of three patients with sporadic Alzheimer's disease. Brain Res. 464:121-131

Weidemann, A., K6ning, G., Bunke, D., Fischer, P., Sal- baum, M.J., Masters, C.L., Beyreuther, K. (1989). Identification, biogenesis and localization of precur- sors of Alzheimer's disease A4 amyloid protein. Cell 57:115-126

Wells, C.E. (1977). Diagnostic evaluation and treatment

in dementia. Dementia, 2nd ed. C.M. Wells, (ed). F.A. Davis, Philadelphia, pp 247-276

Whitson, J.S., Selkoe, D.J., Cotman, C.W. (1989). Amy- loid 13 protein enhances the survival of hippocampal neurons in vitro. Science 243:1488-1490

Whitson, J.S., Glabe, C.G., Shintani, E., Abcar, A., Cot- man, C.W. (1990). 13-Amyloid protein promotes neuritic branching in hippocampal cultures. Neurosci. Lett. 110:31%324

Wion, D., Le Bert, M., Brachet, P. (1988). Messenger RNAs of 13-amyloid precursor protein and prion protein are regulated by nerve growth factor in PC12 cells. Int. J. Dev. Neurosci. 6:387-393

Wirak, D.O., Bayney, R., Ramabhadran, T.V., Fracasso, R.P., Hart, J.T., Hauer, P.E., Hsiau, P., Pekar, S.K., Scangos, G.A., Trapp, B.D., Unterbeck, A.J. (1991). Deposits of amyloid 13 protein in the central nervous system of transgenic mice. Science 253:323-325

Wischik, C.M., Crowther, R.A., Stewart, M., Roth, M. (1985). Subunit structure of paired helical filaments in Alzheimer's disease. J. Cell Biol. 100:1905-1912

Wischik, C.M., Novak, M., Edwards, P.C., Klug, A., Tichelar, A. (1988). Structural characterization of the core of the paired helical filament. Proc. Natl. Acad. Sci. U.S.A. 85:4884--4888

Wisniewski, H.M., Terry, R.D. (1973). Reexamination of the pathogenesis of the senile plaque. Progress in Neu- ropathology. H.M. Zimmerman (ed). Grune and Strat- ton, New York, pp 1-26

Wisniewski, H.M., Kozlowski, P.B. (1982). Evidence for blood-brain barrier changes in senile dementia of the Alzheimer type (SDAT). Ann. N.Y. Acad. Sci. 396:119-189

Wisniewski, H.M., Narang, J.K., Terry, R.D. (1976). Neurofibrillary tangles of paired helical f'daments. J. Neurol. Sci. 27:173-181

Wisniewski, H.M., Moretz, R.C., Lossinsky, A.S. (1981). Evidence for induction of localized amyloid deposits and neuritic plaques by an infectious agent. Ann. Neurol. 10:517-522

Wong, C.W., Quaranta, V., Glenner, G.G. (1985). Neu- ritic plaques and cerebrovascular amyloid in Alzhei- mer disease are antigenically related. Proc. Natl. Acad. Sci. U.S.A. 82:8729-8732

Yamada, T., Sasaki, H., Furuya, H., Miyata, T., Goto, I., Sakaki, Y. (1987). Complementary DNA for the mouse homolog of the human amyloid beta protein precursor. Biochem. Biophys. Res. Commun. 149:665- 671

Yankner, B.A., Villa-Komaroff, L., Neve, R.L. (1988). The biological activity of Alzheimer's amyloid. Soc. Neurosci. 14:896 (abstract)

Yankner, B.A., Dawes, L.R., Fisher, S., Villa-Komaroff, L., Oster-Granite, M.L., Neve, R. (1989). Neurotox- icity of a fragment of the amyloid precursor associated with Alzheimer's disease. Science 245:417--420

Yankner, B.A., Duffy, L.K., Kirschner, D.A. (1990). Neurotrophic and neurotoxic effects of amyloid 13 protein: Reversal by tachykinin neuropeptides. Science 250:27%282


Zain, S.B., Salim, M., Chou, W.-G., Sajdel-Sulkowska, E.M., Majocha, R.E., Marotta, C.A. (1988). Molecu- lar cloning of amyloid cDNA derived from mRNA of the Alzheimer brain. Coding and non-coding regions of the fetal precursor mRNA are expressed in the Alzhei- mer cortex. Proc. Natl. Acad. Sci. U.S.A. 85:92%933

Zain, S.B., Chou, W.G., Majocha, R.E., Ventosa- Michelman, M., Marotta, C.A. (1989). Genetically engineered cells that overexpress amyloid A4 (beta amy-

loid) of Alzheimer disease. American Association of Neuropathologists Annual Meeting abstract 103. J. Neuropathol. Exp. Neurol. 48:335

Zimmerman, K., Herget, T., Salbaum, J.M., Schubert, A., Hilbich, C., Cramer, M., Masters, C.L., Mul- thaup, G., Kang, J., Lamaire, H.G. (1988). Localiza- tion of the putative precursor of Alzheimer's disease- specific amyloid at nuclear envelopes of adult human muscle. EMBO J. 7:367-372

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Molecular and cellular biology of Alzheimer amyloid