Rationale: Amyloidogenic processing of beta-amyloid precursor protein (APP) leading to the formation of beta amyloid (Abeta) is a key event in the pathogenesis of Alzheimer's disease (AD). Abeta is sequentially derived by a cleavage at the residues 671/673 by beta-site APP cleaving enzyme, followed by a cleavage at the residues 711/713 by gamma-site APP cleaving enzyme. Point mutations in APP such as in Swedish double mutations, are known to promote beta-site cleavage. Therefore, we hypothesize that antisense blocking of the mutated beta-site, and not of the normal gamma-site, will reduce translation of amyloidogenic APP-mRNA but not normal APP-mRNA, in a model harboring Swedish mutations (KM670/671NL) (TgAPPSWE or Tg2576). We tested this hypothesis in Tg2576 (TgAPPSWE) transgenic mice by blocking translation of APP-mRNA with 2'-Methoxy-O-Ethyl (MOE)-modified antisense oligodeoxynucleotides (ODNs) directed at the mutated beta-and normal gamma-sites. The data show that 2'MOE modified antisense ODNs directed at the beta-site significantly reduced cerebral levels of soluble Abeta40/42 (sAbeta40/42) while the levels of total detectable alpha-cleaved soluble APP (sAPPalpha) showed a small increase [Siegel and Chauhan, SFN-2002, 687.14]. Antisense blocking at the gamma-site did not produce any effect. These results indicate that beta-site blocking may have arrested the translation of amyloidogenic but not mouse APP, while gamma-site blocking is non-specific. Based on these results, current application proposes the use of a Swedish double mutant model (TgAPPSWE) harboring point mutations in APP (K670M/N671L), and a model overexpressing normal wild type human APP (TgAPPWT) as a control to test the efficacy of antisense ODNs directed at the beta-site of mutated APP in reducing amyloid burden and amyloid-associated toxicity. This project is to test the potential usefulness of antisense ODNs to the beta-site as therapy for familial AD. Hypothesis: Antisense blocking at the mutated beta-site will inhibit the translation of amyloidogenic APP without affecting normal APP processing in TgAPPSWE and TgAPPWT.
Specific Aims : 1. Determine if antisense blocking at the mutated beta-site will reduce the levels of detectable mutated amyloidogenic APP-mRNA without affecting mouse APP-mRNA and production of normal alpha-cleaved APP in TgAPPSWE or normal human and mouse APP-mRNA in TgAPPWT as evaluated by cRT-PCR quantitation of normal and mutant APP-mRNA and ELISA measurement of sAPPalpha. 2. Determine if antisense blocking at the mutated beta-site will reduce cerebral amyloid load in TgAPPSWE, as evaluated by ELISA and immunocytochemistry of Abeta peptides. 3. Determine if antisense blocking at the mutated beta-site will prevent and reverse Abeta-induced synaptotoxicity and the neurotoxic response of microglia and astroglia in TgAPPSWE, as evaluated by immunocytochemistry and imaging analysis of synaptic markers and markers of reactive microglia and astroglia. Significance: Targeting the translation of amyloidogenic APP by antisense synthetic oligodeoxynucleotides may prove to be an effective and safe form of gene therapy for treating familial AD and other genetic diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21NS044538-01A2
Application #
6780661
Study Section
National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
Program Officer
Refolo, Lorenzo
Project Start
2004-04-01
Project End
2006-01-31
Budget Start
2004-04-01
Budget End
2005-01-31
Support Year
1
Fiscal Year
2004
Total Cost
$148,077
Indirect Cost
Name
University of Illinois at Chicago
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Chauhan, Neelima B; Siegel, George J (2007) Antisense inhibition at the beta-secretase-site of beta-amyloid precursor protein reduces cerebral amyloid and acetyl cholinesterase activity in Tg2576. Neuroscience 146:143-51
Chauhan, Neelima B; Sandoval, Jimena (2007) Amelioration of early cognitive deficits by aged garlic extract in Alzheimer's transgenic mice. Phytother Res 21:629-40
Chauhan, Neelima B (2007) Intracerebroventricular passive immunization with anti-oligoAbeta antibody in TgCRND8. J Neurosci Res 85:451-63
Chauhan, Neelima B; Siegel, George J; Feinstein, Douglas L (2005) Propentofylline attenuates tau hyperphosphorylation in Alzheimer's Swedish mutant model Tg2576. Neuropharmacology 48:93-104
Chauhan, Neelima B; Siegel, George J (2005) Efficacy of anti-Abeta antibody isotypes used for intracerebroventricular immunization in TgCRND8. Neurosci Lett 375:143-7
Chauhan, Neelima B; Siegel, George J; Feinstein, Douglas L (2004) Effects of lovastatin and pravastatin on amyloid processing and inflammatory response in TgCRND8 brain. Neurochem Res 29:1897-911
Chauhan, Neelima B; Siegel, George J; Lichtor, Terry (2004) Effect of age on the duration and extent of amyloid plaque reduction and microglial activation after injection of anti-Abeta antibody into the third ventricle of TgCRND8 mice. J Neurosci Res 78:732-41
Chauhan, Neelima B; Siegel, George J (2004) Intracerebroventricular passive immunization in transgenic mouse models of Alzheimer's disease. Expert Rev Vaccines 3:717-25