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

Abstract

Misprocessing of β-amyloid precursor protein (APP) leading to the formation of elevated quantities of β-amyloid peptide (Aβ), derived by a cleavage at the β-secretase site (N-671/673aa) and by a cleavage at the γ-secretase site (C-711/713aa) of APP, is considered a key event in the pathogenesis of Alzheimer disease (AD). Point mutations near the β-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 β-site cleavage and to increase levels of Aβ. Aβ 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 β-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 β-site (AS-β site) or with AS-ODNs directed at the normal γ-site (AS-γ site) of human APP-mRNA, and compared with procedural controls that received i.c.v. injections of sense ODNs at the β-site (S-β site), sense ODNs at the γ-site (S-γ 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 β- and γ-cleaved soluble Aβ40 (sAβ40), β- and γ-cleaved soluble Aβ42 (sAβ42) and α-cleaved soluble β-amyloid precursor protein (sAPPα). 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-β increased cerebral levels of sAPPα by 43% and reduced sAβ40/42 by ∼39%; while simultaneously reducing the cortical density of AChE by ∼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-γ did not have any effect. These results indicate that AS directed to the mutated β-site may be an effective approach to treat familial AD.