Role Of MicroRNA-153 In The Pathogenesis Of Alzheimer's Disease Mouse Model

Alzheimer's disease (AD), the most common form of dementia, is a neurodegenerative disorder, characterized by amyloid plaques and neurofibrillary tangles. With the fast increasing of aging population, incidence of the aging associated diseases especially dementia are sharply increasing, affecting the life quality of old people enormously. However, AD is a multi-factor involved complex disease. so far the pathogenesis is still indefinite. MicroRNAs (miRNAs) are endogenous small non-coding RNAs of～22 nucleotides that act to control gene expression by inhibiting the translation of target transcripts. Increasing evidence show miRNAs play a role in the development of many disease, in which the reports related with AD can also be found. However. the research in this fild is just at the beginning, further explore the miRNAs that deregulated during the disease process and the significance of these changes would not only uncover a new pathway of the pathogenesis but also a new approach of the therapy.Amyloid protein precursor (APP). the precursor of the Aβpeptide. is a crucial risk factor of AD since increased expression of which can lead to overloading of A(3 peptide. APP is a member of a family of closely related transmembraue glycoproteins that includes APLP1 and APLP2 in mammals. A lot studies have confirmed that APLP2 also participate in AD pathogenesis.In this study. we detected the brain miRNA profile of double transgenic APPswe/PSΔE9 murine model, the result indicating that miR-153 was differential expressed. Further analyzed by quantitative PCR. we confirmed that in 3-and 9-month old AD mice, the level of miR-153 is significantly decreased. Predicted by databases, a putative binding site of mir-153 on APP and APLP2 gene 3'UTR was identified respectively. By renilla luciferase activity assay, we validated the functional interaction of these sites. Detected by Western-blot, the APLP2 protein level was showed significantly up-regulated in 3-and 9-month old AD mice brain.To further confirm the tune role of miR-153 on APP and APLP2 expression. the stablly transfected SH-SY5Y cells over-expressng miR-153 were constructed. The level of target genes in these cells were then analysed. The results show that the protein level of APP/APLP2 were clearly reduced. meanwhile. the mRNA abundance of the two genes were also decreased at some level. indicating that mir-153 acts on these targets mainly through inhibition of translation but it can also promote mRNAs degradation. On the other hand. inhibiting the endogenous expression of miR-153 in M17 cells by miRNA inhibitor. the APLP2 protein level was obviously increased. Mir-153 expression vector or negative control plasmid were co-transfected with APP construct which contain full-length 3'UTR into 293T cells.48h later. the expression of APP were analysed. The data suggest that consistent with the results from stably transfected cells. the reduction in APP protein and mRNA were observed in APP and mir-153 transiently contransfected group versus APP and miRNA control contransfected set. In the loss of function experiment. miR-153 and APP plasmid were cotransfected into 293T cells,24h later, the cells were treated with mir-153 inhibitor or inhibitor control, then 48h later the level of APP were determined. The findings indicate that compared with inhibitor control processed group, mir-153 inhibitor treatment can significantly increase APP protein level providing the reverse evidence that mir-153 is a regulator of APP/APLP2.To validate the tune role of miR-153 on APP/APLP2 in vivo. miR-153 transgenic mice and double mutated AppLon/Swe transgenic mice expressing intact APP 3'UTR were generated. Then miR-153/APPLon/Swe positive mice were produced by crossing of these two transgenic mice. By western-blot, the APLP2 protein level in the brain of miR-153 transgenic mice were found clearly decreased as compared with control. Meanwhile, the APP protein level in the brain of miR-153/APPLon/Swe positive mice were analyzed and the data show that APP protein were also reduced at some level versus APPLon/Swe control.To confirm the regulator effect of miR-153 on APP/APLP2 during physiological brain develoment. the expression of miR-153 as well as APP/APLP2 protein and mRNA in the brain of gestational day 14.5 to 12-month old wild-type C57 were determined. The data suggest that during this stage. the expression of miR-153 was negatively correlated with the APP/APLP2 protein level. however. the mRNA abundance of the genes fluctuated mildly relative to the enormous protein change implying that during regular brain development the expression of APP/APLP2 are tuned by miR-153.In view of the important role of A(342 and oxidative stress in the pathogenesis of AD and it was reported that both these two factors can influence the expression of miRNAs. To explore the effect of them on the expressin of mir-153. M17 cells were treated with AP42 peptides and H2O2 respectivly. then at different time point. the level of miR-153 and APLP2 protein were analyzed. The result show that both Aβ42 and H2O2 can affect the level of miR-153 and the change was time dependent. Correspondingly. along with the fluctuation of miR-153. the level of APLP2 protein varied inversely indicating that Aβ42 and oxidative stress are powerful regulator of miR-153 therefore leading to the aberrant expression of it's target genes.In this study we confirm that miR-153 can specificly interact with the 3'UTR of APP/APLP2 regulating their expression suggesting that miR-153 play a role in AD pathogenesis.