Elsevier

Neurobiology of Aging

Volume 23, Issue 3, May–June 2002, Pages 335-348
Neurobiology of Aging

Neurobiology of Aging
FAD mutant PS-1 gene-targeted mice: increased Aβ42 and Aβ deposition without APP overproduction

https://doi.org/10.1016/S0197-4580(01)00330-XGet rights and content

Abstract

To investigate the consequences of mutant presenilin-1 (PS-1) expression under the control of the normal PS-1 gene, a gene-targeted mouse bearing the FAD mutation P264L was made. Gene-targeted models are distinct from transgenic models because the mutant gene is expressed at normal levels, in the absence of the wild-type protein. PS-1P264L/P264L mice had normal expression of PS-1 mRNA, but levels of the N- and C-terminal protein fragments of PS-1 were reduced while levels of the holoprotein were increased. When crossed into Tg(HuAPP695.K670N/M671L)2576 mice, the PS-1P264L mutation accelerated the onset of amyloid (Aβ) deposition in a gene-dosage dependent manner. Tg2576/PS-1P264L/P264L mice also had Aβ deposition that was widely distributed throughout the brain and spinal cord. APPNLh/NLh/PS-1P264L/P264L double gene-targeted mice had elevated levels of Aβ42, sufficient to cause Aβ deposition beginning at 6 months of age. Aβ deposition increased linearly over time in APPNLh/NLh/PS-1P264L/P264L mice, whereas the increase in Tg2576 mice was exponential. The APPNLh/NLh/PS-1P264L/P264L double gene-targeted mouse represents an animal model that exhibits Aβ deposition without overexpression of APP.

Introduction

Although the majority of cases of Alzheimer’s disease (AD) are late onset and sporadic, a small percentage of cases are autosomal dominant with early onset [22], [27], [65]. Chromosome 21 familial AD (FAD) mutations are in the APP gene, which is the precursor of the amyloid plaque component, Aβ [64, for review]. The Swedish mutations at the N-terminus of Aβ lead to elevated production of Aβ40 and Aβ42 [7], [12]. Aβ42, the more fibrillogenic and earliest constituent of amyloid plaques [31], [32], is selectively elevated by mutations at the C-terminus [18], [69]. Mutations in PS-1 and presenilin-2 (PS-2), on chromosomes 14 and 1 respectively, are responsible for the majority of FAD cases [42], [59], [66]. Numerous missense PS-1 mutations [14], [22], in-frame deletions of exon 9 [54], [56], and an intron 4 mutation resulting in an insertion (T113-114ins) [16] have been described. The PS-1 mutations cluster in the transmembrane domains at conserved residues, in the second transmembrane domain and in exon 8, using the exon nomenclature of the Alzheimer’s Disease Collaborative Group [1].

FAD cases with PS-1 mutations have a higher cortical amyloid plaque burden immunoreactive for Aβ42 than sporadic AD cases [20], [30], [38], [43]. Like the C-terminal mutations in APP, the PS-1 and PS-2 mutations selectively elevated Aβ42 in plasma and fibroblasts from FAD cases [15], [63]. Elevated levels of Aβ42 were also found in cells transfected with mutant PS-1 or PS-2 and in mice expressing mutant PS-1 [3], [13], [15], [16], [17], [50], [52]. Although mice with PS-1 mutations have elevated levels of Aβ42, they have not deposited Aβ [3], [4], [11], [13], [17], [52]. When transgenic mice with PS-1 mutations were crossed with transgenic mice bearing the Swedish APP mutations, there was marked acceleration in the formation of Aβ deposits [4], [25], [35].

We have used a gene-targeting approach to generate AD models that does not involve protein overexpression. In one model, both Swedish FAD mutations and additional missense mutations to humanize mouse Aβ were introduced into the mouse APP gene [58]. This mouse (APPNLh/NLh) produced normal levels of APP, overproduced human Aβ40 and Aβ42, but did not deposit Aβ. Here we report the introduction of the P264L mutation into the mouse PS-1 gene. The P264L mutation is a non-conservative amino acid substitution in exon 8, causing an onset of FAD at 34–56 years of age [9], [44], [75]. APPNLh/NLh/PS-1P264L/P264L double gene-targeted mice were generated and had normal levels of APP, but elevated levels of Aβ42 sufficient to cause Aβ deposition. Mice with the PS-1P264L mutation were also crossed with transgenic Tg2576 mice that overexpress human APP695 with the Swedish FAD mutations [26] in order to evaluate the effects of this PS-1 FAD mutation on AD-related pathologies.

Section snippets

Construction of the PS-1 gene-targeting vector

Mouse PS-1 genomic DNA was cloned from a bacteriophage library of 129/Sv mouse DNA [57], using a 477-bp probe homologous to PS-1 cDNA sequences from exons 7 through 11. From 6 independent clones that covered 26.5 kb, exons 7 and 8 were positioned on a restriction map. To generate the P264L mutation and a novel AflII site, 3 base-pair changes were introduced into a 200-bp XbaI-BamHI subclone (position 11.5–11.7) using a two-step PCR mutagenesis strategy (Fig. 1). The primary structure of the

mRNA and protein levels in PS-1P264L/P264L mice

Northern blot analysis demonstrated a 3.1 kb band for PS-1 mRNA. PS-1 mRNA levels were quantified after they were normalized with GAPDH mRNA levels. Removal of the PGK-neo gene resulted in mRNA levels in homozygous (PS-1P264L/P264L) mice that were comparable to those in wild type (PS-1+/+) mice (Fig. 2A and D).

Western blotting demonstrated an N-terminal PS-1 fragment of ∼30 kDa, a C-terminal PS-1 fragment of ∼20 kDa, and PS-1 holoprotein of ∼45 kDa. Blotting with CP160, preabsorbed with

Discussion

We have reported on the generation of a gene-targeted mouse containing the PS-1P264L mutation. This FAD mutation has been shown to elevate levels of Aβ42 and markedly accelerate Aβ deposition when crossed into the Tg2576 mouse. Furthermore, we have described the first genetic animal model of amyloid deposition created in the absence of APP overexpression in the double gene-targeted APPNLh/NLh/PS-1P264L/P264L mouse.

Although the PS-1P264L/P264L mouse, following removal of the PGK-neo cassette,

Acknowledgements

We thank James Hirsch, Brian Steffy, and Veerle Baert for technical assistance and Renée Simmons and Edwin McCabe for their excellent care of the mice.

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    Current address: Pfizer, Inc., Eastern Point Road, Groton, CT 06340.

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    Current address: Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.

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