A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model

Abstract

Alzheimer disease (AD) is characterized by excessive deposition of amyloid beta-peptides (A beta peptides) in the brain. In the nonamyloidogenic pathway, the amyloid precursor protein (APP) is cleaved by the alpha-secretase within the A beta peptide sequence. Proteinases of the ADAM family (adisintegrin and metalloproteinase) are the main candidates as physiologically relevant alpha-secretases, but early lethality of knockout animals prevented a detailed analysis in neuronal cells. To overcome this restriction, we have generated transgenic mice that overexpress either ADAM10 or a catalytically inactive ADAM10 mutant. In this report we show that a moderate neuronal overexpression of ADAM10 in mice transgenic for human APP([V717I]) increased the secretion of the neurotrophic soluble alpha-secretase-released N-terminal APP domain (APPs alpha), reduced the formation of A beta peptides, and prevented their deposition in plaques. Functionally, impaired long-term potentiation and cognitive deficits were alleviated. Expression of mutant catalytically inactive ADAM10 led to an enhancement of the number and size of amyloid plaques in the brains of double-transgenic mice. The results provide the first in vivo evidence for a proteinase of the ADAM family as an alpha-secretase of APP, reveal activation of ADAM10 as a promising therapeutic target, and support the hypothesis that a decrease in alpha-secretase activity contributes to the development of AD.

Figure 1

Characterization of ADAM10 transgenic mice. Immunohistochemical detection of HA-tagged ADAM10 using the antibody Y-11. Cortex (A) and hippocampal (B) sections of ADAM10-mo transgenic animals and a nontransgenic control (FVB/N) are shown. Scale bars: 200 ∝m in A and 100 ∝m in B. (C) Quantitation of catalytically active ADAM10 protein levels. Enriched ADAM10 proteins from adult mouse brains were subjected to Western blot analysis and detected with an anti-ADAM10 antibody. The upper panel shows mature ADAM10 (<m) and the proform of ADAM10 (<p). The mature 62-kDa ADAM10 form was quantified using a ³⁵S-labeled secondary antibody (lower panel).

Figure 2

Detection and quantitation of human APP processing products in double-transgenic ADAM10-mo ∞ APP_[V717I], ADAM10-hi ∞ APP_[V717I], ADAM10-hz ∞ APP_[V717I], and ADAM10-dn ∞ APP_[V717I] mice as well as in monotransgenic APP_[V717I] (APP) animals. All analyzed mice were 18 weeks old. (A) APP processing products detected by Western blot analysis as described in Methods. First row, secreted APPsα; second row, secreted APPsβ; third row, membrane-bound APP C-terminal fragments (CTFα, CTFβ); last row, membrane-bound full-length APP (APP fl). (B) Quantitative analysis of the APP-processing products APPsα, APPsβ, and APP CTFα from different mouse lines. In all experiments, quantified APP processing products were normalized to APP fl expression. Values are expressed as percentages of values from APP control mice (set to 100%) and are the mean ± SD (n = 7_8 animals of each line). Significance was determined by the unpaired Student’s t test. *P < 0.05; ^#P < 0.01; P < 0.001. (C) Quantitation of soluble human Aβ40 and Aβ42 peptides isolated from mouse brains by sandwich ELISA. APP_[V717I] mice (n = 6) were used as control to ADAM10-hz ∞ APP_[V717I] (n = 7) and ADAM10-dn ∞ APP_[V717I] (n = 6) animals. Values are the mean ± SEM of the amount of each Aβ peptide per gram of mouse brain. Significance was determined by the unpaired Student’s t test. *P < 0.05.

Figure 3

Detection and quantitation of amyloid plaques in brains from 17- to 19-month-old (A_D) APP_[V717I] transgenic mice, double-transgenic ADAM10-mo ∞ APP_[V717I] mice, and ADAM10-dn ∞ APP_[V717I] mice. Immunohistochemical detection of amyloid plaques in the neocortex in paraffin-embedded sections with either antibody 6F/3D (A) or antibody 4G8 (B). Note that in ADAM10-mo ∞ APP_[V717I] mice, no additional plaques were detected by antibody 4G8. Arrows point to blood vessels. Scale bars: 200 ∝m. (C and E) Thioflavine S_stained β structures in the subiculum of either 17- to 19-month-old (C) or 12-month-old mice (E); scale bars: 200 ∝m. (D) Quantitation of amyloid load in subiculum in thioflavine S_stained sections obtained from 17- to 19-month-old animals. Surface thioflavine S staining is expressed as a percentage of the total subiculum surface. Statistical analysis was performed for each genotype with the following number of animals: APP_[V717I], n = 5; ADAM10-mo ∞ APP_[V717I], n = 13; ADAM10-dn ∞ APP_[V717I], n = 6. *P < 0.05; **P < 0.01.

Figure 4

Analysis of amyloid deposits in brains from 17- to 19-month-old double-transgenic ADAM10-mo ∞ APP_[V717I] (A and B) and ADAM10-dn ∞ APP_[V717I] mice (C and D). Isolated tiny (A) and diffuse (B) amyloid deposits in the brain of an ADAM10-mo ∞ APP_[V717I] mouse. Immunohistochemistry was performed with antibodies 6F/3D (A) and 4G8 (B), detecting either only Aβ peptides or Aβ peptides in addition to N-terminally truncated Aβ peptides (p3 fragments). Scale bars: 100 ∝m. (C and D) Analysis of amyloid plaque composition in the brain of an ADAM10-dn ∞ APP_[V717I] mouse. Immunohistochemistry was performed with antibodies FCA3542 (C) and FCA3340 (D), detecting peptides containing AβX-42 and AβX-40, respectively. Antibody FCA3340 detects fewer plaques than FCA3542 does. Scale bars: 200 ∝m.

Figure 5

LTP studies of hippocampal slices of transgenic mice. (A) LTP in hippocampal slices. The slope of the field excitatory postsynaptic potential (fEPSP) is plotted as a function of time before and after high-frequency stimulation in slices from APP_[V717I] mice (filled circles, n = 6) and from ADAM10-hz ∞ APP_[V717I] mice (open circles, n = 5). The two arrows indicate the time at which the two tetani were applied. Samples of traces obtained from ADAM10-hz ∞ APP_[V717I] and APP_[V717I] mice are shown at the top. Recordings of fEPSPs obtained either 10 minutes or 1 hour and 30 minutes after tetanic stimulation are superimposed on the same control trace obtained before stimulation. (B) Input-output curve of fEPSP amplitude (mV) versus stimulus (V) at the Schaffer collaterals did not differ significantly between genotypes. (C) Paired-pulse facilitation did not differ between APP_[V717I] (n = 6) and ADAM10-hz ∞ APP_[V717I] (n = 7) mice at the tested interval of 50 ms. Traces showing typical paired-pulse facilitation in an APP_[V717I] and in an ADAM10-hz ∞ APP_[V717I] mouse are shown on the right. Significance was determined by unpaired Student’s t test. *P < 0.01.

Figure 6

Morris water-maze. (A) Acquisition of place learning in the Morris water-maze hidden-platform task. Learning deficit in APP_[V717I] transgenic mice was ameliorated in double-transgenic ADAM10-mo ∞ APP_[V717I] mice. Lines represent mean ± SEM for 9_10 mice per group. *P < 0.05, **P < 0.01, APP_[V717I] vs. control; ^_P < 0.05 APP_[V717I] vs. ADAM10-mo ∞ APP_[V717I] by Student's t test. (B) Memory test in Morris water-maze probe trial without platform. The deficit in APP_[V717I] transgenic mice was improved in double-transgenic ADAM10-mo ∞ APP_[V717I] mice. Bars represent mean ± SEM for 9_10 mice per group. *P < 0.05, APP_[V717I] vs. control; ^_P < 0.05, APP_[V717I] vs. ADAM10-mo ∞ APP_[V717I] by Student's t test.