Synaptotrophic effects of human amyloid β protein precursors in the cortex of transgenic mice

BRAIN RESEARCH ELSEVIER

Brain Research 666 (1994) 151-167

Research report

Synaptotrophic effects of human amyloid/3 protein precursors in the cortex of transgenic mice L. Mucke a,*, E. Masliah b, W.B. Johnson a, M.D. Ruppe a, M. Alford b, E.M. Rockenstein a, S. Forss-Petter c,**, M. Pietropaolo d, M. Mallory b, C.R. Abraham d a

Department of Neuropharmacology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA b Departments of Neurosciences and Pathology, University of California, San Diego, La Jolla, CA 92093-0624, USA c Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA d Departments of Medicine and Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA

Accepted 6 September 1994

Abstract The amyloid precursor protein (APP) is involved in Alzheimer's disease (AD) because its degradation products accumulate abnormally in AD brains and APP mutations are associated with early onset AD. However, its role in health and disease appears to be complex, with different APP derivatives showing either neurotoxic or neurotrophic effects in vitro. To elucidate the effects APP has on the brain in vivo, cDNAs encoding different forms of human APP (hAPP) were placed downstream of the neuron-specific enolase (NSE) promoter. In multiple lines of NSE-hAPP transgenic mice neuronal overexpression of hAPP was accompanied by an increase in the number of synaptophysin immunoreactive (SYN-IR) presynaptic terminals and in the expression of the growth-associated marker GAP-43. In lines expressing moderate levels of hAPP751 or hAPP695, this effect was more prominent in homozygous than in heterozygous transgenic mice. In contrast, a line with several-fold higher levels of hAPP695 expression showed less increase in SYN-IR presynaptic terminals per amount of hAPP expressed than the lower expressor lines and a decrease in synaptotrophic effects in homozygous compared with heterozygous offspring. Transgenic mice (2-24 months of age) showed no evidence for amyloid deposits or neurodegeneration. These findings suggest that APP may be important for the formation/maintenance of synapses in vivo and that its synaptotrophic effects may be critically dependent on the expression levels of different APP isoforms. Alterations in APP expression, processing or function could contribute to the synaptic pathology seen in AD. Keywords: Alzheimer's disease; Amyioid precursor protein; Mutation; Transgenic; Synapse; Neuroplasticity

1. Introduction The severe progressive dementia of Alzheimer's disease (AD) is at this point in time still incurable. It is emotionally devastating to both victims and their families and, because of its duration and increasing prevalence [32], presents an enormous socio-economic problem. At the structural level, the cognitive impairment of A D correlates most closely with a loss of neuronal synapses in the frontal cortex [81]. Other features characteristically found in A D brains include amyloid plaques, congophilic angiopathy, neurofibrillary tangles

* Corresponding author. Fax: (1) (619) 554-9981. ** Present address: Research Unit for Experimental Neuropathology, Austrian Academyfor Sciences, A-1090Vienna, Austria. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01074-9

and loss of neurons [66]. While a variety of environmental and genetic factors have been postulated to play a role in the development of A D [9,26,48,94], the etiology of A D remains unknown. Several recent observations have called attention to the involvement of amyloid proteins, particularly the amyloid precursor protein (APP), in the pathogenesis of AD. Several different isoforms of A P P are derived from a single A P P gene by alternative splicing [50], including the 695 amino acid form (APP695) which is expressed primarily in neurons [51,92] and the 751 amino acid form (APP751) which contains a domain that is strongly homologous to the Kunitz type family of serine protease inhibitors and has been shown to function as a protease inhibitor [27,52,56,74,80,85]. Within the nervous system, A P P is found primarily in

152

L. Mucke et al./ Brain Research 666 (1994) 151-167

neurons [4,7,71] with a preferential localization at central and peripheral synaptic sites [64]. Proteolytic degradation of APP gives rise to the A/3 protein, a 39-43 amino acid fragment which constitutes an important component of amyloid plaques [15,24,43,65,67, 70,72]. Notably, point mutations that alter the amino acid sequence of APP near the region of the Aft protein are strongly associated with early onset familial AD (FAD) (for review see [8]). While many studies suggest that alterations in APP processing can result in increased amyloid formation and neurodegeneration [17,49,67], a number of observations seem inconsistent with this 'amyloid hypothesis' [59,81]. To understand the role APP plays in AD, it is essential to define its primary function(s). Several studies indicate that APP derivatives can be either neurotoxic or neuroprotective/neurotrophic in vitro (see [45] for review). A potential role for APP in neuroplasticity is also suggested by in vivo studies which identified APP in growing neurites of immature rat brain [39] and demonstrated that APP reaches its highest level of expression in the CNS at the time of brain maturation and completion of synaptic connections [33]. The current study aimed to assess the effects of wildtype and mutated human APPs on the intact CNS by expressing them in neurons of transgenic mice. We report that neuronal overexpression of APP increased the number of synaptophysin immunoreactive presynaptic terminals in the frontal cortex of transgenic mice but, over the course of 24 months, did not lead to amyloid deposition or neurodegeneration. These findings indicate that APP or its derivatives may play a key role in synaptic plasticity in vivo and that processes which interfere with APP function could contribute to the loss of synapses seen in AD.

2. Materials and methods 2.1. Construction of transgenes The cDNAs encoding the 695 and 751 amino acid forms of human APP (hAPP695, hAPP751) were a generous gift from Dr. D. Goldgaber (Department of Psychiatry, SUNY, Stony Brook, NY). The C to T missense mutation [16] was introduced into the 162 bp 5'BgllI-Styl3' segment of the hAPP cDNAs by PCR primer modification. Two oligonucleotide primers were synthesized: LMAPPI: 5 ' - d A A G A C G G A G G A G A T C T C T G A - 3 ' , and LMAPP2: 5'dATCACCAAGGTGATGATGAT-3'. Primer LMAPP2 introduced the GTC[Val] ~ ATC[IIe] mutation. Primers were used at a final concentration of 0.4/xM in a standard PCR mix to amplify 800 ng of the circular APP695 plasmid using 2.5 U Taq polymerase and a Perkin Elmer Cetus thermal cycler (Model 9600). Thermoprofile: 5 min 94°C initial denaturation; 30 cycles with 1 min 94°C, 1 min 45°C, 1 min 72°C; 5 min 72°C final extension. The resulting amplicon was subcloned in pCR1000 (TA cloning kit from InVitrogen, San Diego, CA) to enable verification of its content by sequence analysis using a Sequenase kit (USB, Cleveland, OH). The mutated BgllI-StyI segment was then used to replace the corresponding wiidtype sequence

in the hAPP695 and hAPP751 cDNAs. Mutated and non-mutated hAPP cDNAs were adapted for insertion into the NSE vector with HindllI linkers. All subcloning steps were carried out according to standard protocols [62]. After completion of the constructs, the correctness of strategic transgene elements including mutated versus wildtype BglII-StyI segments and NSE-hAPP junctions were confirmed by sequence analysis,

2.2. DNA probes For hybridizations of slot blots, Southern blots and Northern blots, the following DNAs were 32p-labeled with a Prime-ItT M II random primer labeling kit (Stratagene, La Jolla, CA): (I) a 215 bp segment of polylinker and SV40 late gene sequence located immediately upstream of the SV40 polyadenylation signal in NSE-hAPP constructs. This probe is transgene-specific and allows differentiation of NSE-hAPP DNAs and mRNAs from endogenous mouse genes and their products. (2) A murine APP-specific probe was generated by PCR amplification of mouse genomic DNA using primers LMAPP3: 5'-ccgatcgaTTTTGTCCACGTATCTTTG-3' and LMAPP4: 5'-ccgaagcTTTATGTAATACAGTGTAGA-3' (capitalized nucleotides represent hAPP sequence). The resulting 198 bp amplicon of murine APP sequence corresponds to a sequence (nucleotides 2752-2935 in [24]) in the 3' untranslated region of hAPP cDNAs which was not incorporated into NSE-hAPP transgenes. Hence, this probe hybridizes with endogenous mouse APP mRNAs but not with transgene-derived message. (3) Approximately 2.8 kb Kpnl/HindllI segment of the hAPP751 cDNA which crosshybridizes extensively with both mouse and human APP coding sequences. (4) B-actin coding sequence (bp 480-bp 740) [83].

2.3. Generation and screening of transgenic mice Male and female B6× SJL mice of different ages (3 weeks to 24 months of age) were used in this study. Animals were maintained in conditions consistent with AAALAC regulations throughout the course of the investigation. Analgesics and anesthetics were used as recommended by the veterinary staff. Transgenes were linearized and freed from plasmid sequences, electrophoresed through a 0.6% SeaPlaque agarose gel (FMC BioProducts, Rockland, ME), passaged over an Elutip-D column (Schleicher & Schuell, Keene, NH) and dialyzed extensively against injection buffer (0.1 mM EDTA/5 mM Tris, pH 7.5). For microinjection, the concentration of transgenes was adjusted to 1.5/zg/ml. Preparation of mice as well as microinjection and reimplantation of one cell stage B6xSJL F2 embryos was carried out according to standard procedures [19]. Transgenic offspring was identifed by slot blot or Southern blot analysis of genomic DNA extracted from tail biopsies. Unless specified otherwise, the transgenic mice included in this study were heterozygous for the NSE-hAPP transgenes, resulting from crosses between a heterozygous transgenic mouse with a non-transgenic B6XSJL F1 breeder. Normal controls consisted of non-transgenic littermates derived from the same crosses. Crosses between heterozygous transgenic mice from a given transgenic line were used to generate homozygous transgenic mice containing a double number of transgene copies compared with their singly transgenic parents. The homozygous state of these mice was confirmed by a progeny test.

2. 4. Northern blot analysis Organs were removed immediately after sacrifice and snap frozen in liquid nitrogen. Poly(A)+-enriched RNA was obtained as described [5] using oligo-dT-cellulose from InVitrogen (San Diego, CA). The amount of RNA obtained from each sample was determined spectrophotometrically. Denatured RNA was separated on 1% agarose-formaldehyde gels, transferred onto Biotrans nylon

L. Mucke et al. / Brain Research 666 (1994) 151-167

DNA. Samples were then spun at 15,800× g for 1 min followed by electrophoresis of supernatants through 7.5% or 14% SDS-polyacrylamide gels. Equal loading of samples was assured by weighing all frozen tissue samples with an accuracy of +0.1 mg and adding precisely 5 volumes of homogenization buffer per sample weight. After transfer of gels onto PDVF membranes (Millipore, Lexington, MA) and blocking of membranes in 5% milk proteins, blots were immunostained with ctC5 or other antibodies that recognize APP or APP derivatives (see Table 2). Binding of primary antibodies was detected with alkaline phosphatase-conjugated secondary antibodies (Promega, Madison, WI) or the ECL method from Amersham (Arlington Heights, IL). For quantitative analyses, brain proteins were extracted as described previously [20] using 100 mg of frozen tissue per sample. To assure equal loading, the protein content of all samples was determined by Lowry assay and adjusted with homogenization buffer to 1.25 mg/ml; 40/zg of brain protein were loaded per lane. Radioiodinated protein A (ICN, Irvine, CA) bound to primary antibodies (for rabbit polyclonals) or secondary antibodies (for primary mouse monoclonals) was detected using a Phosphorimager SF (Molecular Dynamics, Sunnyvale, CA). Signals were quantitated by integrating pixel intensities over defined volumes using the ImageQuant software. Quantitative comparisons with signals obtained from defined amounts of recombinant APP (kindly provided

membranes (pore size: 0.2 /~m) from ICN (Irvine, CA), UV crosslinked and hybridized sequentially with the SV40, APP and actin probes. Hybridizations were carried out between 60° and 70°C in 50 mM PIPES, 50 mM sodium phosphate, 50 mM NaCI, 1 mM EDTA and 5% SDS for 16 h. Washes were done at the hybridization temperature with 0.2×SSC/5% SDS for 20-40 min. In between hybridizations with different probes, blots were stripped of the preceding probe with 95°C water containing 0.2% SDS. Blots were exposed to Hyperfilm-MP (Amersham, Arlington Heights, IL) for 4 h to 3 days. Signals obtained with the actin probe were used to correct for RNA loading errors. Radioactive signals were quantitated by integrating pixel intensities over defined volumes using a Phosphorimager SF (Molecular Dynamics, Sunnyvale, CA) and the ImageQuant software. 2.5. Western blot analysis Snap frozen brain tissue was homogenized in 5 volumes of lysis buffer (1 mM EGTA, 1 mM EDTA, 1 mM AEBSF [4-(2 aminoethyl)-benzenesulfonylfluoride), 1 mM leupeptin, 1% Triton X-100, in PBS), spun at 300× g for 30 min, mixed with 5 vols. of 1 × sample buffer [30], boiled for 1 min and sonicated to destroy

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Fig. 1. Structure and identification of NSE-hAPP transgenes. A: diagrammatic illustration of NSE-hAPP constructs. Mutated and non-mutated cDNAs encoding full-length hAPP695 or hAPP751 were placed under the regulatory control of sequences derived from the rat neuron-specific enolase (NSE) gene, including approximately 2.8 kb of the 5' flanking region, exon 1 (El), intron 1 (I1) and 6 bp of exon 2 (for structure of NSE gene, see [61]). The polyadenylation signal at the 3' end of the construct is provided by SV40 late gene sequence. B: slot blot analysis of genomic DNA extracted from tail biopsies of NSE-hAPP transgenic and non-transgenic mice. The slot blot was hybridized with a 32p-labeled probe that recognizes the SV40 sequence at the 3' end of the NSE-hAPP construct (see A and Materials and Methods). Serial dilutions of a plasmid containing the NSE-hAPP751 construct added to genomic DNA from non-transgenic mice were used as positive controls and to estimate transgene copy numbers. C: detection of mutated versus non-mutated NSE-hAPP transgenes in genomic DNA by PCR and BclI digest. Genomic DNA was amplified by PCR using primer a (5'-AAGACGGAGGAGATCTCTGA-3') located 165 bp upstream, and primer b (5'-AACGGGTrTGTTI'CTTCCCA-3') located 264 bp downstream of the C to T mutation associated with familial AD [16]. No amplification products were seen in non-transgenic mice because the primer sequences are located within the two exons that flank the mutated exon 17 on either side and amplification across two introns was ineffective. Because the C to T mutation generates a BclI site which is lacking in the wildtype sequence, amplification products were digested with this restriction enzyme. Amplicon cleavage by BclI identifies transgenic mice as carrying mutated NSE-hAPP transgenes.

L. Mucke et al. / Brain Research 666 (1994) 151-167

154

by R. Siman and R. Scott, Cephalon, Inc.) were used to estimate the amount of hAPP in nanogram per milligram of brain protein extract.

2.6. Immuno-histopathological analysis and in situ quantitation of hAPP and GAP-43 expression Mice were anesthetized by intraperitoneal injection of 0.5 ml of a 3.8% chloral hydrate solution and flush-perfused transcardially with normal saline. Brains were removed immediately and postfixed either with 4% paraformaldehyde/0.25% glutaraldehyde in PBS (pH 7.4) at 4°C for 48 h or with 70% ethanol/0.15 M NaCI at room temperature for 24-48 h. Prior to further processing, specimens were assigned code numbers (by W.B.J.) to ensure objective assessment. Codes were broken only after the analysis was complete. Paraformaldehyde-fixed brains were serially sectioned at 4 0 / z m with a Vibratome 2000. Free-floating sagittal sections were immunolabeled for microdensitometrical analysis with the Quantimet 570C and for confocal imaging (see below). Ethanol-fixed brains were embedded in paraffin and sectioned at 9 / z m . Ethanol-fixed sections were blocked with 5% dry non-fat milk in TBST (10 m M Tris (pH 8), 150 m M NaCI, 0.05% Tween 20) and then labeled with the primary antibodies listed in Table 2 (diluted 1:100 to 1:1000 in TBST). Binding of primary antibodies was detected with species-matched biotinylated secondary antibodies and revealed with avidin-biotin peroxidase kits (Vector, Burlingame, CA) using diaminobenzidine ( D A B ) / H 2 0 2 as substrates. Sections were stained for amyloid with Congo red and thioflavin S. To enhance the staining of potential

amyloid deposits, ethanol- and paraformaldehyde-fixed sections were also treated with formic acid for various time periods prior to immunostaining with the 1280 and the Yoshiko antibodies (Table 2). Transgenic brains were compared with age- and strain-matched non-transgenic control brains (usually from non-transgenic littermates) by detailed visual inspection of serial microscopic sections. Relative levels of neuronal hAPP and GAP-43 expression were determined in situ with the Quantimet 570C as described [36]. Briefly, optical density values were measured in select fields of ethanol-fixed brain sections immunolabeled with a C 5 and vibratome sections immunolabeled with anti-GAP43. Corrected optical density values were obtained by subtracting background optical densities (from sections immunostained in the absence of primary antibodies). The Q u a n t i m e t 570C was also used, as described previously [90], to determine neuronal counts in brain sections stained with 1% Cresyl violet. Statistical analyses of the results were carried out with the STAT V I E W II (Abacus Concepts, Berkeley, CA) software package.

2. 7. Confocal microscopy and computer-aided quantitation of CNS alterations Forty micrometer thick paraformaldehyde-fixed brain sections were double immunolabeled as described [34] using different combinations of antibodies directed against (i) hAPP, (ii) the synaptic marker synaptophysin, (iii) the dendritic marker MAP-2, a n d / o r (iv) the growth-cone associated marker GAP-43 (see Table 2). Sections

Table 1 Summary of transgenic mice analyzed in this study Transgene Constructs

Individual Transgenic Founders

Bred into Tg Line

NSE-hAPP695

NSE-hAPP695-17 NSE-hAPP695-31 NSE-hAPP695-39 NSE-hAPP695-40 NSE-hAPP695-60 NSE-hAPP695m-1 NSE-hAPP695m-2 NSE-hAPP695m-19 NSE-hAPP695m-21 NSE-hAPP695m-25 NSE-hAPP695m-28 NSE-hAPP695m-36 NSE-hAPP695m-40 NSE-hAPP751-24 NSE-hAPP751-28 NSE-hAPP751-35 NSE-hAPP75 lm-3 NSE-hAPP751m-8 NSE-hAPP751m-16 NSE-hAPP751m-21 NSE-hAPP751m-32 NSE-hAPP751m-55 NSE-hAPP751m-57 NSE-hAPP751m-58

No * Yes Yes No * Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No * Yes No * Yes Yes Yes Yes ** Yes No *

NSE-hAPP695m

NSE-hAPP751

NSE-hAPP751 m

Tg Copy No.

1 1

10 1

10 4

WB n

IH n

2 4

2

10 2 2 13 2 10 2 1 2 12 16

5

15 12

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2

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Age range of mice analyzed: 3 weeks-24 months. Abbreviations: IH = Immunohistochemical staining of ethanol-fixed brain sections with t~C5. m = C to T mutation [16]. n = n u m b e r of mice analyzed. NP = neuropathoiogical evaluation for amyloid formation and neurodegeneration (see Table 2 and text for details). S R P T = synaptophysin immunoreactive presynaptic terminals. Tg = transgenic. WB = Immunostaining of Western blots with a C 5 (see Table 2). + through + + + + + = level of hAPP expression as determined by semiquantitative analysis of Western blots immunostained with aC5. * = breeding not pursued because of mosaicism. ** = line could not be maintained due to premature death of F1 offspring (cause unknown). Empty cells/fields = not analyzed or does not apply.

L. Mucke et al. / Brain Research 666 (1994) 151-167 were then incubated with a mixture of FITC-conjugated horse antimouse IgG and biotinylated goat anti-rabbit IgG secondary antibodies, followed by Avidin D Texas red (Vector, Burlingame, CA). The double-labeled sections were transferred to SuperFrost plus slides (Fisher, Tustin, CA) and mounted under glass coverslips with antifading media containing 4% n-propyl gallate (Sigma, St. Louis, MO). For each mouse, three serial sections of corresponding brain regions were examined using the Bio-Rad MRC-600 laser scanning microscope [34,35] mounted on an Axiovert Zeiss microscope. Digitized images (4/section/case), 0.5 micrometer in thickness, were transferred to a Macintosh Ilci, running the public domain program of Wayne Rasband (Image 1.43) [35,36]. The area of the neuropil occupied by MAP-2 immunolabeled dendrites was quantified and expressed as a percentage of the total image area, as described previously [34]. Thresholded synaptophysin immunoreactive elements were counted automatically as described [35] and expressed as number of presynaptic terminals per unit area (100 /zm2). Previous studies in experimental animal models of denervation and reinnervation have confirmed the validity of this approach [36,87]. In addition, correlations of values obtained by laser scanning confocal microscopy with data obtained by electron microscopic analysis [37] and dot blot immunoquantitation [2] have corroborated the linearity of the results obtained by confocal microscopy. Because recent studies have stressed the importance of obtaining nonbiased 3-dimensional (3-D) information on particle numbers and size for the accurate quantita-

tion of neurons and synapses [12], it is critical to note that our quantitative data on presynaptic terminals and neuronal dendrites were generated from real 3-D reconstructions derived from serial laser scanning confocal microscopic optical sections as described previously [35]. Statistical analyses of the results were conducted as outlined above. Confocal images of double-immunolabeled sections were also inspected for morphoiogic alterations of neuronal cell bodies and dendrites.

3. Results

3.1. Generation of NSE-hAPP transgenic mice The rat neuron-specific enolase promoter has previously been shown to target the expression of fusion genes to neurons in vivo [13]. To assess the effects different isoforms of human APP (hAPP) have on the intact CNS, cDNAs encoding full-length hAPP695 or hAPP751 (see Materials and Methods) were placed downstream of NSE regulatory sequences (Fig. 1A). To

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Fig. 2. Brain expression of endogenous mAPP and transgene-derived hAPP mRNAs. Poly(A)+-enriched RNA from the brains of transgenic mice and non-transgenic controls was analyzed on a Northern blot by sequential hybridization with different DNA probes (see Materials and Methods). The following groups of mice were compared: Four- to nine-month-old heterozygous ( + ) or homozygous (+ + ) transgenic (Tg) mice from lines NSE-hAPP751m-57 and NSE-hAPP695m-19 and age-matched six-month-old non-transgenic (Non-Tg) controls. A: autoradiograph obtained by exposure of the Northern blot to X-ray film following hybridization with the probes indicated on the left. B-D: quantitation of hybridization signals using a phosphorimager (see Materials and Methods). X-axis: numbers 1-8 correspond to the different samples of brain RNA indicated in A. Y-axis: hybridization signals (integrated pixel intensities). B: SV40 probe specific for transgene-derived message. C: APP cDNA probe which recognizes both mouse and human APP transcripts. D: mAPP probe which recognizes endogenous mouse APP transcripts but does not recognize transgene-derived mRNA. In this analysis only signals obtained with the same probe were compared quantitatively. Signal intensities obtained with different probes cannot be compared directly due to differences in the size and GC content of the probes used. Note that the level of hAPP mRNA in brain was approximately twice as high in homozygous-transgenic mice than in heterozygous-transgenic mice from the same line (compare samples 1/3 with sample 2 and samples 4/5 with sample 6 in A and B). See text for further details.

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L. Mucke et a l . / Brain Research 666 (1994) 151-167

evaluate the in vivo effects of the Val to lie change at position 717 (in APP770), which is linked with early onset familial AD [16], the C to T mutation encoding this amino acid substitution was introduced into the hAPP695 and hAPP751 cDNAs by PCR primer modification. The fusion genes resulting from the ligation of non-mutated/wildtype and mutated hAPP cDNAs with the NSE promoter were microinjected individually into B6 x SJL F2 one cell stage embryos. Twenty four founder mice transgenic for NSEhAPP695 (n = 5), NSE-hAPP695m (n = 8) (m = mutated), NSE-hAPP751 (n = 3) or NSE-hAPP751m (n = 8) were identified by slot blot analysis of genomic DNA (Fig. 1B) using a probe that recognizes the SV40 element of the NSE-hAPP fusion genes (Fig. 1A). From 19 of these founder mice, transgenic lines could be established (Table 1). While transgene copy numbers varied across lines from 1 to approximately 10, transmission of transgenes from parents to offspring within individual lines was stable with respect to transgene copy numbers and integration sites over at least 5 generations, as determined by Southern blot analysis

(data not shown). The identity and genetic consistency of lines was also ensured by random selection of mice from every generation and line and confirmation of the presence or absence of the C to T mutation in their genomic DNA by PCR/restriction endonuclease analysis (Fig. 1C). From five of the founders, transgenic lines could not be established, primarily due to mosaicism (Table 1).

3.2. Neuronal expression of hAPP in NSE-hAPP transgenic mice Cerebral transgene expression was readily detectable at the mRNA level in the majority of transgenic lines by Northern blot analysis using a probe that recognizes the SV40 sequence which provides the polyadenylation signal for NSE-hAPP-derived transcripts (Figs. 1A, 2A and B). Within the same line, brain hAPP mRNA levels in transgenic homozygous mice were roughly twice as high as those in transgenic heterozygous mice (Fig. 2A and B). To determine differences in the level of APP transcripts in brains of

Table 2 Antibodies used for labeling of Western blots and brain sections Target Antigen

Antibody

Type

Source

Reference

A/31-40 A/31-38 A/31-28 A/31-28 amyloid, plaque amyloid, plaque amyloid, vascul. amyloid + NFTs amyloid + NFTs hAPP500-648 APP1-695 APP1-695 APP secreted APP secreted APP676-695 APP586-606 APP649-672 APP666-695 APP676-695 APP18-38 APP N-term. APP 666-695 APP 676-695 APP 676-695 APP KPI APP KPI GAP-43 GFAP MAP-2 neurofilament synaptophysin Tau

R1280 Yoshiko Rl152 Lily Angela Corina Flora Shira Varda aC5 LN21 LN27 PN2 P 2-1 R2293 AB10 10 E1 12 E1 385C 63N 22C11 61C C-7 C-8 KPI SY KPI BVN 5 G-A-5 AP20 SMI312 SY38 Alz 50

rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal mouse monoclonal mouse monoclonal rabbit polyclonal mouse monoclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal mouse monoclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal mouse monoclonal mouse monoclonal mouse monoclonal mouse monoclonal mouse monoclonal

D. Selkoe D. Selkoe Cephalon Inc. D. Selkoe D. Selkoe D. Selkoe D. Selkoe D. Selkoe D. Selkoe Athena Neurosciences J. Trojanowski J. Trojanowski W. Van Nostrand W. Van Nostrand C. Abraham E. Koo Cephalon Inc. Cephalon Inc. Cephalon Inc. H. Yamaguchi Boehringer H. Yamaguchi D. Selkoe D. Selkoe S. Younkin W. Van Nostrand J.Baudier Boehringer Boehringer Sternberger Inc. Boehringer Abbot Laboratories

[79] [22] [63] [69] [68] [69] [21] [1] [1] [52,53] [4] [4] [84] [85] unpublished [14] [63] [63] [73] [93] [29] [93] [55] [55] [54] unpublished [40] [82] [82] [41] [40] [91]

Abbreviations: GFAP = glial fibrillary acidic protein. KPI = Kunitz type protease inhibitor domain. MAP = microtubule associated protein. NFTs = Neurofibrillary tangles. APP amino acid residues numbered according to APP695 except for aC5 (numbering based on APP751).

L. Mucke et al. / Brain Research 666 (1994) 151-167

transgenic and non-transgenic mice, brain RNA was analyzed on Northern blots using a probe which crosshybridizes with murine and human APP coding sequences. Overall APP mRNA levels in brains of homozygous transgenic mice from the highest expressor line were found to be approximately 5-fold higher than those found in brains of non-transgenic controls (Fig. 2C). Notably, neuronal overexpression of hAPP in transgenic mice did not induce an aberrant increase or compensatory decrease in the expression of the endogenous murine APP gene. Hybridization of RNA samples with a probe which recognizes endogenous murine APP transcripts, but not transgene-derived mRNA, revealed similar mAPP mRNA levels in transgenic and non-transgenic mice (Fig. 2D). hAPP protein was identified and differentiated from mouse APP (mAPP) by Western blot analyses of brain homogenates and immunostaining of ethanol-fixed brain sections using the aC5 antibody (see Table 2 and Materials and Methods). Under the conditions used here, aC5 readily detected hAPP but showed only minimal cross-reactivity with endogenous mAPP (Figs. 3 and 4). For each line of NSE-hAPP transgenic mice, 1-26 individuals from the F1 or subsequent generations were screened for cerebral hAPP expression (Table 1). The mice included in this expression screen were 3 weeks-24 months of age. Offspring from 17 of the 24 transgenic founder mice showed clear neuronal expression of hAPP (Table 1 and Fig. 4C). The distribution of transgene expression in the NSE-hAPP transgenic mice closely resembled the expression pattern previously described for other NSE-driven fusion genes [13]. A similar widespread neuronal hAPP expression was found in all expressor lines analyzed (Table 1); it consistently included both neocortex and hippocampal formation, two regions prominently involved in AD. A mouse from a high expressor line was used here to illustrate the typical distribution of NSE-driven hAPP expression (Fig. 4A). Endogenous APP has been shown to be transferred from cell body to nerve endings by fast anterograde axonal transport [29,64]. Within transgenic neurons, hAPP immunoreactivity was found in the cell soma as well as in presynaptic terminal boutons (data not shown), indicating that hAPP expressed in central mouse neurons also undergoes anterograde axonal transport. Levels of hAPP expression varied across separate transgenic lines (Table 1, Figs. 3 and 4), most likely due to differences in the transgene integration sites within the genome of the founders from which the separate transgenic lines were derived. However, within any given transgenic line CNS/neuronal hAPP levels were similar in mice from different generations (F1-4) and age groups (2-13 months) (Figs. 3 and 4). In brains from all trangenic expressor lines, the highest levels of

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Fig. 3. hAPP protein expression in brain homogenates of NSE-hAPP transgenic mice. Brain homogenates from transgenic and non-transgenic mice were analyzed on Western blots using antibodies raised against human APP (aC5, see Table 2) as described in Materials and Methods. A: typical staining pattern found in mice transgenic for NSE-hAPP751 constructs (lane 1) compared with mice transgenic for NSE-APP695 constructs (lane 2) and non-transgenic (Non-Tg) controls (lane 3). The multiple bands in lanes 1 and 2 probably represent differently glycosylated a n d / o r truncated APP molecules of the same isoform. B: comparison of hAPP expression across mice from different transgenic lines (see also Table 1). C: Comparison of hAPP expression in mice of different ages from the same line (NSEhAPP695m-19). Note the consistency of expression levels over time and across different mice derived from the same line. rhAPP = recombinant full-length human APP751 (50 ng). w = week(s), m = months.

hAPP immunoreactivity were observed in the frontal cortex (Fig. 4A and E). For all four NSE-hAPP constructs, differences in hAPP protein expression across separate transgenic lines paralleled steady state hAPP mRNA levels, i.e. lines with high levels of transgenederived message had higher hAPP protein levels than lines with low hAPP mRNA levels. Across lines, there was no correlation between transgene copy number and level of transgene expression (Table 1). For each of the four NSE-hAPP constructs, two expressor lines were selected for a more detailed analy-

158

L. Mucke et al. / Brain Research 666 (1994) 151-167

25% (61C) to 34% (22Cll) of the total amount of APP expressed in the cortex. It is interesting that the increase in total APP expression in transgenic mice over non-transgenic control levels was higher at the RNA than at the protein level (compare Fig. 2C with Fig. 5B) as this difference may reflect post-transcriptional mechanisms that counteract overproduction of APP at the protein level.

sis. For comparisons of mutated and non-mutated hAPPs of the same isoform (either 695 or 751), lines with roughly similar levels of cerebral hAPP expression were used (Figs. 3B, 4F). This approach controls for the possibilities that the expression of human proteins affects mouse neurons non-specifically and that biological differences between lines could be due to differences in neuronal protein load. Brain levels of total APP (hAPP plus mAPP) were compared between transgenic and non-transgenic mice on Western blots using antibodies which cross-react with hAPP and mAPP (Fig. 5). Based on this analysis, it is estimated that in transgenic heterozygotes from our highest NSE-hAPP expressor line (NSE-hAPP695m-19), hAPP695m expression increased brain APP levels to approximately 133% (61C) to 152% (22Cll) compared with non-transgenic controls (100%) (Fig. 5B). Hence, in NSE-hAPP heterozygous mice from this transgenic line the mutated human APP may make up roughly

3.3. Increase in the number of synaptophysin immunoreactive (SYN-IR) presynaptic terminals in the frontal cortex of NSE-hAPP transgenic mice Because in vitro studies have suggested that APP or some of its breakdown products may fulfill neurotrophic functions (see [45] for review), the relationship between the number of presynaptic terminals and the expression of growth-associated proteins (GAP-43 and APP) was studied quantitatively in six of the trans-

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L. Mucke et al. /Brain Research 666 (1994) 151-167

genic lines (Table 1). Brain sections from transgenic mice and non-transgenic controls were double-immunolabeled for hAPP and synaptophysin (a marker of presynaptic terminal boutons [42]) or for hAPP and GAP-43 (a growth-cone associated marker [36]) and studied with laser scanning confocal microscopy/computer aided image analysis as described previously [36]. Initially, non-transgenic mice were assessed to de-

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termine the influence of the genetic background on the number of synaptophysin immunoreactive (SYN-IR) presynaptic terminals. For this purpose, counts of cortical SYN-IR presynaptic terminals in the frontal region were compared among C57BL/6 and SJL inbred mice and C57BL/6 x SJL (B6 x SJL) F1 hybrids (4 mice analyzed per strain). Average numbers ( + S.E.M.) of SYN-iR presynaptic terminals per 100 /zm 2 were: 93 ( + 3) for C57BL/6, 93 ( + 3) for SJL, and 89 (+ 7) for B6 x SJL. These differences were not statistically significant by one-factor ANOVA, indicating that the inheritance of variable proportions of C57BL/6 or SJL genome by transgenic mice derived from (B6 x SJL) x (B6 x SJL) crosses should not significantly influence the number of SYN-IR presynaptic terminals. The number of SYN-IR presynaptic terminals was then compared across different brain regions among different lines of NSE-hAPP transgenic mice (Table 1) and normal non-transgenic littermate controls, Transgenic mice from lines with moderate levels of hAPP expression showed a statistically significant increase in the number of SYN-IR presynaptic terminals in their frontal cortices compared with non-transgenic controls (Fig. 6A-C, F and G). Compared with non-transgenic controls, transgenic mice from line NSE-hAPP751m-57 also displayed a significant increase in synaptophysin immunoreactivity by dot blot analysis/phosphorimager quantification of brain homogenates (5 mice/line, P = 0.01 by unpaired two-tailed Student's t-test), carried out as described previously [2] (data not shown).

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L. Mucke et al. /Brain Research 666 (1994) 151-167

160

When comparing lines matched for similar levels of neuronal hAPP expression, there was no significant difference in the effect of mutated versus non-mutated forms of hAPP on the number of SYN-IR presynaptic terminals (Fig. 6A and B). The average intensity of SYN-IR per presynaptic terminal and average area of individual SYN-IR presynaptic terminal boutons was assessed in the frontal cortex of transgenic and non-transgenic mice by thresholding images of synaptophysin immunolabeled brain sections as described [35]. This analysis revealed that the difference in the average intensity of SYN-IR expressed per presynaptic terminal in transgenic mice (mean pixel intensity = 194.5 + 2.5 (S.E.M.); 3 mice analyzed for each of the 5 lines shown in Fig. 6) and non-transgenic controls (mean pixel intensity = 191 +

4.7 (S.E.M.); 3 mice analyzed) was not statistically significant ( P > 0.4 by Mann-Whitney U-test). The average size (+ S.E.M.) of SYN-IR presynaptic terminal boutons in these transgenic (0.341 + 0.013 /xm 2) and non-transgenic mice (0.343 + 0.049/zm 2) also did not differ significantly (P > 0.8 by Mann-Whitney Utest). When NSE-APP751 transgenic mice (n = 15 in total) from four separate lines ( - 2 4 , -28, m-21, m-57) were compared by quantitative immunocytochemistry of brain sections, a positive correlation was identified in the frontal cortex between levels of hAPP expression and number of SYN-IR presynaptic terminals in twomonth-old (r = 0.71, P < 0.01) and in eleven-month-old (r = 0.66, P < 0.01) mice. In contrast, in the hippocampus, midseptum, cerebellum and basal ganglia no sta-

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L. Mucke et al. / Brain Research 666 (1994) 151-167 tistically significant differences in S Y N - I R p r e s y n a p t i c t e r m i n a l c o u n t s were observed b e t w e e n t r a n s g e n i c a n d n o n - t r a n s g e n i c mice (Fig. 6C). All h A P P - p o s i t i v e p r e s y n a p t i c t e r m i n a l s also expressed G A P - 4 3 i m m u n o r e a c t i v i t y (not shown); G A P 43 is c o n s i d e r e d a m a r k e r of n e u r i t i c growth a n d plasticity (see [40] for review). Across different lines of N S E - h A P P 7 5 1 t r a n s g e n i c mice, a strong c o r r e l a t i o n was f o u n d b e t w e e n the n u m b e r of S Y N - I R p r e s y n a p t i c t e r m i n a l s in the f r o n t a l cortex a n d expression of G A P 43 ( r = 0.85, P < 0.002, n = 11). I n contrast, cell c o u n t s in all areas of the b r a i n analyzed, i n c l u d i n g the frontal cortex, were similar in t r a n s g e n i c a n d n o n - t r a n s g e n i c mice (Fig. 6D) a n d t r a n s g e n i c mice showed n o decrease in the width of their cerebral cortices (data n o t shown). I n line N S E - h A P P 6 9 5 m - 1 9 , which showed higher n e u r o n a l levels of h A P P expression t h a n any of the o t h e r lines (Figs. 2 - 5 , T a b l e 1), the increase in cortical S Y N - I R p r e s y n a p t i c t e r m i n a l c o u n t s per a m o u n t of h A P P expressed was smaller t h a n in lines expressing hAPP751 at lower levels (Fig. 6A, B a n d E). T o determ i n e w h e t h e r this difference reflects a g e n e r a l difference in the s y n a p t o t r o p h i c p o t e n t i a l of APP751 versus APP695, t r a n s g e n i c mice from t h r e e a d d i t i o n a l low a n d m o d e r a t e h A P P 6 9 5 expressor lines were e x a m i n e d : at low to m o d e r a t e levels of expression, h A P P 6 9 5 also showed significant s y n a p t o t r o p h i c effects, w h e r e a s at higher levels of expression t h e r e was a progressive decline in s y n a p t o t r o p h i c effects (Fig. 7). T o f u r t h e r evaluate the dose effect of h A P P expression, we comp a r e d N S E - h A P P heterozygotes a n d homozygotes from

161

individual t r a n s g e n i c lines. I n c r e a s e d h A P P expression in homozygous mice from low a n d m o d e r a t e expressor lines i n c r e a s e d the cortical expression of synaptophysin a n d G A P - 4 3 , w h e r e a s f u r t h e r elevation of h A P P levels in homozygotes from the highest expressor line NSEh A P P 6 9 5 m - 1 9 led to a decrease in s y n a p t o t r o p h i c effects (Fig. 8).

3.4. L a c k o f amyloid deposits or neurodegeneration T o d e t e r m i n e w h e t h e r A P P overexpression in this m o d e l i n d u c e s amyloid f o r m a t i o n or n e u r o d e g e n e r a tion, e t h a n o l - or p a r a f o r m a l d e h y d e - f i x e d b r a i n sections from 2- to 2 4 - m o n t h - o l d t r a n s g e n i c a n d n o n - t r a n s g e n i c mice ( T a b l e 1) were s t a i n e d with a n t i b o d i e s which readily detect amyloid deposits, n e u r i t i c a b n o r m a l i t i e s , n e u r o f i b r i l l a r y tangles or gliosis in b r a i n s of A D patients (Table 2). I n addition, n e u r o n a l c o u n t s were d e t e r m i n e d in b r a i n sections s t a i n e d with Cresyl violet using the Q u a n t i m e t 570C a n d the integrity of n e u r o n a l d e n d r i t e s was assessed in b r a i n sections i m m u n o l a b e l e d with the d e n d r i t i c m a r k e r M A P - 2 by laser s c a n n i n g confocal microscopy a n d c o m p u t e r - a i d e d statistical analysis. T h e s e m e t h o d s were used b e c a u s e they have previously allowed us to q u a n t i t a t e different degrees of n e u r o d e g e n e r a t i o n in t r a n s g e n i c mice expressing the g p l 2 0 e n v e l o p e p r o t e i n of HIV-1 [82]. T h e histopathological analysis i n c l u d e d a 13-month-old homozygous t r a n s g e n i c m o u s e from the highest h A P P expressor line ( N S E - h A P P 6 9 5 m - 1 9 ) as well as the 17m o n t h - o l d f o u n d e r m o u s e N S E - h A P P 7 5 1 m - 5 5 , the highest expressor of the h A P P 7 5 1 isoform. No deposi-

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Fig. 6. Quantitation of synaptophysin immunoreactive presynaptic terminals and neurons. Brain sections from transgenic mice and age-matched non-transgenic controls were either immunolabeled with antibodies against the presynaptic marker synaptophysin (SYN) or stained with Cresyl violet. Computer-aided statistical analysis of images generated by laser scanning confocal microscopy was used to quantitate the number of SYN-IR presynaptic terminals (see Materials and Methods for details). A-B: percent increase in the number of SYN-IR presynaptic terminals in 5 separate lines of transgenic mice at 2 months (A) and 11 months (B) of age over the average number of SYN-IR presynaptic terminals (arbitrarily defined as 100%) found in age-matched non-transgenic controls (6 mice analyzed per group and time point). Statistically significant increases (by unpaired two-tailed Student's t-test) are indicated by asterisks ** P < 0.007, * P < 0.025, (*) P < 0.05]. Note that similar relative increases in synaptic counts were found in transgenic mice at both time points analyzed. Compare the differences in the number of SYN-IR presynaptic presynaptic terminal across separate transgenic lines with the differences in hAPP expression shown in Fig. 4F. C: comparison of SYN-IR presynaptic terminal counts in different brain regions between ll-month-old transgenic mice from line NSE-APP751m-57(n = 9) and age-matched non-transgenic controls (n = 31). Statistically significant differences were found only within the frontal cortex. Compare these findings with the typical differences in the levels of hAPP expression across different brain regions shown in Fig. 4F. ** P < 0.01 by unpaired two-tailed Student's t-test. D: cell counts within the frontal cortex, obtained using the Quantimet 570C on brain sections stained with Cresyl violet, did not differ significantlybetween NSE-hAPP751m-57transgenic (n = 13) and non-transgenic (n = 13) mice. Previous studies have shown that in sections of non-gliotic brains, cells ranging in size from 25 to over 100 /.tm2 were immunoreactive with neuronal markers, whereas cells under 25 /zm2 were labeled with astroglial markers [90]. E: hAPP effects were further compared in 2- and 11-month-old mice from lines NSE-hAPP751m-57 and NSE-hAPP695m-19 (n = 3 per line and age) by dividing the percent increase in the number of cortical SYN-IR presynaptic terminals (see A and B above) by the amount of hAPP immunoreactivity expressed in corresponding regions of the opposite hemispheres (see Fig. 4F). The resulting ratio indicates the percent increase in the number of SYN-IR presynaptic terminals achieved by one optical density unit of hAPP immunoreactivity (Synaptotrophic Effect). The difference between the two lines was significant (P < 0.01 by unpaired two-tailed Student's t-test). F,G: laser scanning confocal images (contrast inverted) of synaptophysin immunolabeled frontal cortex sections from a non-transgenic control (F) and a hAPP751 transgenic mouse of the moderate expressor line 57 (G). Punctate structures (in black) represent SYN-IR nerve terminals, large rounded blank spaces neuronal cell bodies. Scale bar = 10/zm. The images shown were derived from individual optical sections (0.2 /zm in thickness). Note that the actual reconstruction and quantitation of SYN-IR presynaptic terminals was based on the computer-aided analysis of serial optical sections (see Materials and Methods for details).

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tions of amyloid or convincing evidence for neurodegeneration were found in any of the mice. Laser confocal image analysis of brain sections immunolabeled with antibodies against MAP-2 (dendritic marker) or SMI312 (axonal marker) (see Table 2) revealed no statistically significant changes in the area of neuropil occupied by neuronal dendrites or axons (data not shown). Furthermore, immunostaining of brain sections with antibodies against GFAP revealed no reactive astrocytosis; reactive astrocytosis is a sensitive indicator of neural injury from diverse causes [11]. Granular clusters of glia-associated fibrillar material that cross-reacted with a variety of polyclonal antibodies

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4. Discussion

In several independent lines of transgenic mice, neuronal overexpression of hAPP increased the number of SYN-IR presynaptic terminals in the CNS. Because transgenic mice showed no significant increases

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163

L. Mucke et aL /Brain Research 666 (1994) 151-167

in the average area of S Y N - I R presynaptic terminal boutons or in the average intensity of synaptophysin immunostaining per presynaptic terminal, it is likely that the increased number of SYN-IR elements in transgenic mice reflects an actual increase in the number of presynaptic terminal boutons rather than an increase in synaptophysin expressed by individual presynaptic vesicles or in the number of S Y N - I R vesicles per presynaptic terminal. The restriction of statistically significant increases in S Y N - I R presynaptic terminals to the frontal cortex raises the question whether neurons in the frontal cortex respond differently to hAPP overexpression than neurons in other brain regions. Alternatively, the topographical restriction of hAPP-induced changes could reflect primarily an hAPP dose effect, since the other brain regions also showed lower levels of hAPP expression than the frontal cortex (Fig. 4A and E). Across low and moderate expressor lines of transgenic mice, hAPP levels correlated positively both with the number of S Y N - I R presynaptic terminals and with the levels of cortical synaptophysin/GAP-43 expression. In addition, increasing the level of hAPP expression within low and moderate expressor lines by generating homozygous offspring clearly increased synaptotrophic effects (Fig. 8). Taken together, these results

provide strong in vivo support for a cause-effect relationship between the overexpression of hAPP and the increase in the number of S Y N - I R and GAP-43 I R presynaptic terminals. It is interesting that at higher levels of hAPP695 expression there was a decline in synaptotrophic effects (Figs. 6E, 7 and 8). These findings raise the question whether the dose response curve for hAPP-induced synaptotrophic effects might be bell-shaped, i.e. beyond a certain level, neuronal overexpression of hAPP may become less effective at increasing the number of SYN-IR presynaptic terminals. While overexpression of full-length hAPPs induced degeneration in cultured transfected embryonal carcinoma P19 cells, differentiated into neurons by exposure to retinoic acid [95], overexpression of hAPP in mature neurons of the intact CNS in the current study did not induce neurodegeneration. However, in independent experiments involving the NSE-hAPP transgenic mice described above, moderate levels of neuronal hAPP751 expression effectively protected the CNS against excitotoxic injuries, whereas several-fold higher levels of hAPP695 expression were not neuroprotective [47] or impaired neuroregeneration [38]. Based on these data, it is possible that elevating hAPP expression levels significantly above those found in our 50

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-/+ +/+

-/+ +/+

-/+ +/+

NSE-hAPP695m-19 ~ NSE-hAPP751rn-57 ~ NSE-hAPP751-28 -/+ Tg Heterozygous +/+ Tg Homozygous

Fig. 8. Comparison of synaptotrophic effects in hAPP transgenic heterozygous ( + / - ) and homozygous ( + / + ) mice from different lines. Proteins were extracted from frontal cortices and analyzed by quantitative Western blot for hAPP levels and by immuno-dotblot for synaptophysin and GAP-43 levels as outlined in Fig. 7. Columns represent results obtained in individual mice. Note the increase in hAPP expression in homozygotes compared with heterozygotes in all three transgenic lines and the substantially higher levels of hAPP expression in line 19 compared with the other two lines. While synaptotrophic effects increased with increased hAPP expression in homozygotesof the low and moderate expressor lines 28 and 57, the increase in hAPP levels in the homozygote from the high expressor line 19 was associated with a decrease in synaptotrophic effects. This decrease in synaptotrophic effects in transgenic homozygotes from line 19 was confirmed in an independent experiment (not shown).

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line 19 would be detrimental to neurons and induce neurodegeneration. Constitutive overexpression of hAPP in transgenic brains failed to induce the formation of amyloid deposits. In another transgenic model, NSE-driven neuronal expression of non-mutated hAPP751 was reported to induce small deposits in the CNS which stained with A/3-cross-reactive antibodies [18,58]. However, the interpretation of these divergent findings is difficult since transgenes were expressed in different strains of mice and different antibodies were used for the histopathological characterization of the two models. For a review of findings obtained in other transgenic models pertinent to AD see [57]. As recently discussed in detail [31], several factors could be responsible for the absence of AD-type pathology in APPtransgenic mice. Evidence is increasing that the pathogenetic effects of FAD-associated APP point mutations involve altered APP processing and increased formation of potentially neurotoxic amyloid (see [9] and [78] for review). However, it has also been speculated that APP point mutations may contribute to the development of AD by decreasing APP neuroprotective functions [59]. Notably, the 717 Val to lie mutation, which is associated with rare cases of FAD [16], did not significantly decrease the synaptotrophic effects of hAPPs in vivo when mutated and non-mutated hAPP751 isoforms were compared in transgenic lines with similar levels of cerebral hAPP expression (Fig. 6A and B). However, as APPs likely fulfill a range of functions [8,45], it is possible that the 717 Val to lie change affects an aspect of APP functioning that was not tested for in our investigation. It is also conceivable that subtle differences in the functioning of mutated versus nonmutated hAPPs were obscured in the CNS of transgenic mice by the activities of endogenous mouse APPs. While APP knockout mice reconstituted with human APPs could, in principle, help assess this issue, it should be noted that two recently identified gene products, APLP1 and APLP2 [86,88,89], show strong homology to APP and, hence, may also be able to compensate for a lack of functional APP. The number of SYN-IR presynaptic terminals in the neocortex appears to be regulated by a variety of growth-associated factors, extracellular matrix proteins, adhesion molecules, proteases and protease inhibitors. The absence of neurodegenerative changes in NSEhAPP transgenic mice indicates that the increase in SYN-IR presynaptic terminals did not represent a compensatory response to denervation, a phenomenon observed in experimental lesion models [3,10,36,44]. In vitro studies suggest that APP or APP derivatives may regulate neurite outgrowth and synaptogenesis via interactions with neuronal adhesion molecules and extracellular matrix proteins [28,75,76] and through effects

on neuronal calcium homeostasis [25,45,46]. It is possible that overexpression of hAPP in vivo increases synapse formation or decreases the synapse drop out which usually follows the period of maximal synapse formation during development [6,77]. In either case, our results are consistent with recent studies which showed that infusion of an APP 17mer peptide into the lateral ventricles induces synaptotrophic effects in the neocortex of aged rats [60]. Taken together, these observations indicate that APPs may play a key role in synaptic plasticity a n d / o r homeostasis in vivo. Processes that alter the expression, metabolism or function of APPs could contribute to the synaptic pathology seen in AD and, hence, form important potential targets for therapeutic interventions.

Acknowledgments We are grateful to the many investigators who have generously contributed antibodies and other materials to this study (see Table 2 and text). We also thank F. Bloom for helpful discussions of our data and E. Picard and J. Price for excellent technical assistance. This work was supported by NIH Grants AG-04342 (L.M.), AG-11385 (L.M.), AG-05131 (E.M.), AG-10689 (E.M.), AG-00001 (C.R.A.) and AG-09905 (C.R.A), by the Alzheimer's Association (FSA-91-010 (L.M.); IIRG-89-125 (C.R.A.)) and by the Department of Health Services, State of California (92-1539 (E.M.)).

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