Protective Effect Of A Novel ABAD-DP Aptamer Against Intracellular Aβ Toxicity

Alzheimer’s disease (AD) is a common neurodegenerative disease,characterized by progressive cognition impairment. So far, there is not anyeffective method for prevention or treatment of AD. AD is the main cause ofdementia. The global prevalence was estimated that35.6million people sufferedfrom dementia in2010, and the numbers of dementia patients were expected toalmost double every20years, to65.7million in2030and115.4million in2050[1]. The pathological hallmarks of AD are extracellular senile plagues,intracellular neurofibrillary tangles and the loss of neurons and synapses.Amyloid peptide (A) is the main component of senile plague. There areextensive evidence showing that A is the key factor involved in the causationand progression of AD. Therefore, A may serve as an effective therapeutictarget of AD.Mitochondrial dysfunction is associated with age-related neurodegenerativediseases. Mitochondrial dysfunction occurs in early stages of AD, as manifestedby energy dysmetabolism, increased free radicals, and intracellular calciumoverload. It has been shown that A can induce mitochondrial structureabnormalities and dysfunction, which plays an important role in the progressionof AD. Mitochondrial A accumulation was found in the brains of AD patientsand mutant amyloid precursor protein (mAPP) transgenic mice. A can functionat the outer membrane, inner membrane, intermembrane space and matrix toexert toxicity through various targets of mitochondria. Recent studies demonstrated that the interaction between A and amyloid binding alcoholdehydrogenase (ABAD) in mitochondria results in accumulation of harmfulintermediate metabolites, which worsen the mitochondrial injury. ABAD decoypeptide (DP), the92-120amino acids of the ABAD LDloop, contains anA-binding site. ABAD-DP can competitively antagonize binding betweenABAD and A, therefore protect ABAD activity and inhibit A-inducedcytotoxicity. ABAD-DP may be a potential therapeutic agent for AD treatment.However, it remains a question on the synthesis of ABAD-DP with stableintracellular expression and high binding efficiency to A.Based on peptide aptamer, the cDNA encoding ABAD-DP was inserted intothe activated site of the human thioredoxin (TRX) to construct theTRX-ABAD-DP-TRX (T-A-T) fusion gene, and adeno-associated virus wasused to allow the stable expression of T-A-T with molecular biological technique.Then we test the effect of T-A-T aptmer on intracellular A toxicity in vitro,aiming to explore a new method for AD treatment. This study was divided intotwo parts:Part1Construction of recombinant adeno-associated virusexpressing TRX1-ABAD-DP-TRX2aptamerObjective: To construct the recombinant adeno-associated virus (rAAV)expressing TRX1-ABAD-DP-TRX2aptamer using molecular biologicaltechnique. Methods:①The target gene fragments TRX1, ABAD-DP and TRX2were obtained by PCR. All the nucleotide sequences were determined using the Sanger chain termination method. Restriction enzyme digestion was alsoperformed to test the construction of these target gene fragments. Then thesetarget gene fragments were subcloned into the multiple clone site of theadeno-associated virus shuttle plasmid pSSHG-CMV to obtain the T-A-T fusiongene. The recombinant plasmid pSSHG/T-A-T was digested with EcoRI andBamHI.②Polyethyleneimine was used to mediate the three plasmid(pSSHG/T-A-T, pAAV/Ad and pHelper) to transfect HeLa cells. Therecombinant adeno-associated virus rAAV/T-A-T was retrieved after transfectionfor72hours. Titers of the rAAV/T-A-T virus were determined using thereal-time quantitative PCR.③NIH-3T3cells were transduced withrAAV/T-A-T. Immunofluorescent staining was performed to test the expressionof T-A-T. Results:①The sequencing results showed that all the nucleotidesequences of TRX1, ABAD-DP and TRX2were consistent with our design. Thesequencing length of TRX1, ABAD-DP and TRX2was114bp,93bp, and235bp, respectively. The T-A-T fusion gene was435bp after restriction enzymedigestion, consistent with the expected size.②The titer of rAAV/T-A-T was3.45×10~8/ml.③After transduction of NIH-3T3cells with rAAV/T-A-T,immunofluorescent staining showed that more than90%of cells displayed greenfluorescence. Conclusions：①The T-A-T aptamer was constructed successfully.②The recombinant adeno-associated virus expressing T-A-T aptamer(rAAV/T-A-T) was established successfully. Part2Protective effect of recombinant adeno-associated virusrAAV/T-A-T against intracellular A toxicityObjective: To test the protective effect of recombinant adeno-associatedvirus rAAV/T-A-T against intracellular A toxicity by using the cell model ofAD. Methods:①The AD cell model was established by packaging therecombinant retrovirus rL42SN, which contained the A42gene.Immunofluorescent staining was performed to test the expression of A42.②NIH-3T3cells were transduced with rAAV/T-A-T and retrovirus rL42SN.Immunofluorescent staining was performed to detect the localization of T-A-Tand A42in NIH-3T3cells.③SH-SY5Y cells were used to test the effect ofrAAV/T-A-T against intracellular A toxicity. Virus rAAV/T-A-T and rL42SNwere added to SH-SY5Y cells according to the four groups: normal control,T-A-T, A and T-A-T plus A. After incubation for72hours, we observed themorphological changes and performed the MTT assay. Results:①Aftertransduction of NIH-3T3cells with rL42SN, immunofluorescent stainingshowed that more than90%of cells displayed red fluorescence.②Immunofluorescent staining showed the co-localization of the greenfluorescence and red fluorescence in the cytoplasm of NIH-3T3cells.③SH-SY5Y cells of T-A-T group and normal control group exhibited normalmorphology with high density. In the A group, SH-SY5Y cells were in badcondition, with low density and a large number of apoptotic and necrotic cells.The cell condition in the T-A-T plus A group was significantly better than that in the A group.④MTT results showed that SH-SY5Y cell viability wassignificantly reduced in the A group when compared with the normal controlgroup (p <0.05). There was no significant difference in cell viability betweenthe T-A-T and normal control groups (p>0.05). Cell viability was significantlyenhanced in the T-A-T plus A group when compared with the A group (p <0.05). Conclusions：①The recombinant retrovirus rL42SN expressing A42wasestablished successfully.②The T-A-T aptamer can bind with A42withinNIH-3T3cells.③The T-A-T aptamer can protect SH-SY5Y cells againstintracellular A42toxicity.