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 AChE activity in vitro. We wished to test whether or not 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 intracerebroventricularly (ICV) 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 ICV 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 ELISA analysis of sAβ40, sAβ42 and sAPPα. The physiological relevance of antisense ODNs was tested by evaluating the cerebral distribution of acetyl cholinesterase (AChE) before and after the treatment. AChE was found increased about 5-fold in Tg cortex as compared to control brain. Results show that compared to 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 ~4-fold in the treated Tg animals, almost to the level found in the control brain (all values p<0.0001, ANOVA, unpaired 2-tailed Student t-test), while AS-γ did not have any effect. These results indicate that antisense directed to the mutated β-site may be an effective approach to treat familial AD.

Experimental Induction of Type 2 Diabetes in Aging-Accelerated Mice Triggered Alzheimer-Like Pathology and Memory Deficits

“…Quantitation of soluble (s) species of cerebral Aβ from HF- or LF-fed SAMP8 mice and HF- or LF-fed SAMPR1 mice was performed using ELISA as established [54–58]. An aliquot containing 100 μg/100μl protein was used to measure sAβ 40 and sAβ 42 (Covance), cytochrome c oxidase, and pyruvate dehydrogenase (Abcam.com) with the use of commercial kits.…”

Antisense oligonucleotides in therapy for neurodegenerative disorders

“…In such case specific lowering of the mutation-containing protein is desired. Selective RNase Hmediated degradation can be achieved using antisense oligonucleotide gapmers targeting (1) specific point mutations [110], (2) structural differences between wild-type and mutant mRNA [111][112][113], or…”

“…For instance, point mutations near the β-secretase site in the human gene for APP lead to a dominantly inherited form of Alzheimer disease [160]. In a transgenic mouse model of Alzheimer disease containing this mutation, translation of the APP mRNA was blocked by MOE-PS gapmer antisense oligonucleotides that bind specifically to the mutated β-secretase site [110]. Repeated injections into the third ventricle (once a week for 4 weeks) reduced the levels of toxic Aβ, indicating that this could be a possible strategy to treat familial Alzheimer disease [110].…”

Antisense oligonucleotide therapeutics for inherited neurodegenerative diseases

“…Mice transgenic for the Swedish mutation have decreased levels of sAPPa, increased Ab, and a subsequent 5-fold increase in cortical acetylcholinesterase (AChE), which is the enzyme responsible for terminating cholinergic signaling. A series of 4 weekly ICV injections of an ASO targeting the mutated b site increased levels of sAPPa by 43%, decreased levels of Ab by 39%, and reduced cortical AChE to nearly wild type levels [66]. Analysis, which did not include evaluation of behavioral phenotypes, was performed 1 week after the final injection.…”