Antisense inhibition at the beta-secretase-site of beta-amyloid precursor protein reduces cerebral amyloid and acetyl cholinesterase activity in Tg2576

Abstract

Misprocessing of beta-amyloid precursor protein (APP) leading to the formation of elevated quantities of beta-amyloid peptide (Abeta), derived by a cleavage at the beta-secretase site (N-671/673aa) and by a cleavage at the gamma-secretase site (C-711/713aa) of APP, is considered a key event in the pathogenesis of Alzheimer disease (AD). Point mutations near the beta-secretase site in the human gene for APP, such as in the Swedish mutation-KM670/671NL, lead to a form of dominantly inherited AD. These mutations are known to promote beta-site cleavage and to increase levels of Abeta. Abeta has been shown previously to increase acetyl cholinesterase (AChE) activity in vitro. We wished to test whether translational blocking of APP-mRNA at the mutated beta-site by antisense (AS) oligodeoxynucleotides (ODNs) directed to the mutated site will reduce cerebral amyloid in the Swedish transgenic mouse model (Tg2576). Mice were injected i.c.v. with AS-ODNs directed at the mutated beta-site (AS-beta site) or with AS-ODNs directed at the normal gamma-site (AS-gamma site) of human APP-mRNA, and compared with procedural controls that received i.c.v. injections of sense ODNs at the beta-site (S-beta site), sense ODNs at the gamma-site (S-gamma site) or mismatched ODNs, and with untreated littermates (Lt) and untreated transgenic mice (Tgs). ODNs were injected into the 3rd ventricle once a week for 4 weeks. Brains were processed for enzyme-linked immunosorbent assay analysis of beta- and gamma-cleaved soluble Abeta40 (sAbeta40), beta- and gamma-cleaved soluble Abeta42 (sAbeta42) and alpha-cleaved soluble beta-amyloid precursor protein (sAPPalpha). The physiological relevance of AS ODNs was tested by evaluating the cerebral distribution of AChE before and after the treatment. AChE was found increased about fivefold in Tg cortex as compared with control brain. Results show that compared with untreated and procedural controls, AS-beta increased cerebral levels of sAPPalpha by 43% and reduced sAbeta40/42 by approximately 39%; while simultaneously reducing the cortical density of AChE by approximately fourfold in the treated Tg animals, almost to the level found in the control brain (all values P<0.0001, analysis of variance, unpaired two-tailed Student's t-test), while AS-gamma did not have any effect. These results indicate that AS directed to the mutated beta-site may be an effective approach to treat familial AD.

Fig. 1

Proteolytic cleavage sites on APP molecule showing the location of Swedish mutation (Blue letters). Arrows indicate antisense oligonucleotide constructs designed to target ISEVKMDAE amino acids (aa) (β-site) and GVVIATVIV aa (γ-site) on APP.

Fig. 2

Effect of β-secretase-site directed antisense inhibition of human APP-mRNA on the levels of sAPPα, sAβ40 and sAβ42 as measured by ELISA (pg/mg protein) in Tg2576 brain. Data are statistically analyzed by ANOVA, unpaired two-tailed Student t-test, and are presented as group means and standard deviation (SD) derived from the average of individual values (n=3) for each group (N=5).

Fig. 3

Distribution of AChE within the dendritic and axonal neuropil of transgenic Tg2576, wild type and non-transgenic littermate brains. Untreated Tgs (Fig. 3A), vehicle-injected Tgs (Fig. 3B), littermate (Fig. 3C), wild type (Fig. 3D), Tgs injected with sense γ-site ODNs (Fig. 3E), Tgs injected with antisense γ-site ODNs (Fig. 3F), Tgs injected with sense β-site ODNs (Fig. 3G), Tgs injected with antisense β-site ODNs (Fig. 3H). Scale bar=30 μm.