Research On The Neuroprotective Effect Of The Recombinant Adeno-associated Virus System Fusing With ABAD-DP

Alzheimer's disease (AD) is the most common etiology of dementia in theelderly and causes gradual, relentless cognitive deterioration resulting in memory lossas well as other behavioral changes. It is characterized by the presence of fourpathologic lesions: senile plaques (SP), neurofibrillary tangles (NFTs), hippocampalpyramidal cellgranulovacuolar degeneration and neurons loss. The epidemiologicstudy showed that estimated35.6million people worldwide had Alzheimer's diseaseand other dementias (AD/D). The total costs associated with dementia are$604billion(2010US dollars), or approximately1%of the world's gross domestic product.Consequently,to find out the effective treatment of AD is a tremendous challenge forneuroscientists.In recent years, mitochondrial dysfunction has been proposed to play a pivotalrole in neurodegenerative diseases, including Alzheimer's disease. Aβ binding alcoholdehydrogenase (ABAD) is a short chain alcohol dehydrogenase in the mitochondrialmatrix, which involved in multiple aspects of metabolic homeostasis. It is importantfor the protective effect under homeostatic conditions and in response to stress.However, research indicates that binding of Aβ to ABAD distorts the enzyme'sstructure, disorders the roles in metabolic homeostasis related to energy metabolismand catabolism of isoleucine and branched-chain fatty acids, leads to mitochondrialdysfunction and cell death, evidenced by increased reactive oxygen species generation,mitochondrial membrane permeability formation and caspase-3-like activity induction,and decreased activities of the Krebs cycle. These effects can be blocked byintracellular transduction of the ABAD decoy peptide(ABAD-DP). However, due toits short half-life and high synthesis costs, ABAD-DP is difficult to be clinically administered as therapeutic agent. Here, we have fused human neurotrophin-4(NT4)and human immunodeficiency virus1-transactivator (Tat) protein to the5' ofABAD-DP cDNA, harvested the recombinant adeno-associated system fusing withABAD-DP, and did research on its neuroprotective effect by the cytotoxic effect ofAβ42and hydrogen peroxide, then the protective effects of rAAV were tested in themAPP transgenic mice. It may provide a promising candidate for the treatment of AD.The main contents are organized as the following:The fusion gene, NT4-TAT-6His-ABAD-DP (NTA), was constructed usingmolecular biology methods. The rAAV/NTA was obtained by co-transfection ofpSSHG/NTA with pGF140and pAAV/Ad into mammalian HEK293cell line usingCa₃(PO₄)₂method. Hela cells were transfected with NTA, Aβ or both of them byrecombinant virus. The expression of NTA, Aβ₄₂or both of them were detected byimmunofluorescence. SH-SY5Y cells were divided into normal control group, H₂O₂group, AAV vector+H₂O₂group and rAAV/NTA+H₂O₂group to observe themorphological changes under inverted phase contrastmicroscope. The cellularproliferation activity and apoptosis were detected by MTT and Annexin V/PI flowcytometry respectively. The APP695transgenic mice were given rAAV/NTA byintranasal administration for5days. The memory function was measured by Morriswater maze test.The experimental result showed that The fusion gene was identified by therestriction enzymes and DNA sequencing. The rAAV/NT4-TAT-6His-ABAD-DP wasconfirmed stable expression of ABAD-DP, and NTA could bind Aβ42peptide effectivein cells. H₂O₂group exhibited cytotoxicity effects and apoptosis. The rAAV/NTAsignificantly decreased neurotoxicity of H₂O₂and the rate of apoptosis in vitro. Dataindicated that rAAV/NTA had the ability of neuroprotective effect not only by bind Aβ,but also as a non-specificity mitochondria protective agent. The animal experimentshowed that intranasal administration of rAAV/NTA had ameliorated memorydysfunction in Morris water maze test. In brief, the recombinant adeno-associatedvirus system fusing with ABAD-DP could provide a promising candidate for the treatment of neurodegenerative diseases induced by mitochondrial dysfunction,including Alzheimer's disease.