SAMP8 Mouse Hippocampus Aberrant Expression Of MiRNA Identification And Preliminary Functional Studies

Alzheimer's disease(AD) is one of the most common dementia in aged people all over the world. AD is a progressive neurodegenerative disorders in the brain of adult mammals occurs throughout life, and the patients have deterioration of memory, cognition, behavior, emotion, and intellect. With great loss of neuron, there is no remarkable cure for this disorder.The Aβpeptides accumulating in the brain of AD patients result from proteolytic cleavage of APP byβ-andγ-secretases. Increased expression of APP is associated with the pathogenesis of Alzheimer's disease. Indeed, gene duplications of the app locus on chromosome 21 is believed to cause AD pathology. Moreover, polymorphisms in the APP promotor that increase transcription have been associated with AD. Thus, understanding the mechanisms that regulate APP expression is relevant for our understanding of the pathogenesis of AD. Studies in neurodegenerative disorders implied some miRNAs showed different profiles and may have connection with such complicated disease.we choose senescence accelerated mouse (SAM) as animal model to study the mechanisms of AD. One of these strains, the SAMP8 (P8), was found to have age-related deficits in learning and memory which has an average life-span of 18.9 months and have been considered as a useful model for studying spontaneous age-related dementia. SAMR1, normal senescenced, is used as the control. Firstly, we used the Morris water maze test and shuttle box/step through test to indentify this model and it showed a stronger learning and memory ability in SAMR1 than SAMP8 mouse of 4-month,8-month and 12-month. And we find that APP protein level is significantly higher in SAMP8 mouse than SAMR1 mouse, however, its mRNA level shows a less higher than the changes in protein level, suggesting the existence of a post-transcriptional regulatory mechanism in charge of APP protein expression.Based on the above results we assume that APP protein expression regulated by miRNA, combined with bioinformatics and biological experiments to find the possible miRNA targeting APP. First of all, we use TargetScan to analyze APP 3'UTR to find the possible miRNAs involved in this biological process, combined the results of miRNA chip data which shows the expressional differences of miRNAs between the hippocampus of SAMP8 and SAMR1 mouse. Since SAMP8 mouse has a higher expressional level of APP protein than in SAMR1 mouse, we choose the lower expressional miRNAs in SMAP8 compared to the control SAMR1 mouse. Then we use the real-time PCR to validate the filtration results. The selected microRNAs, miR-16, miR-144, miR-195, miR-383 have a follow-up analysis of luciferase activity and protein level authentication, the changes of miRNA-16 is the most significant.It showes a great conservation in the analysis of the miR-16 target sites of APP 3'UTR between different species using miRanda. The mutation of the target site has been introduced in the luciferase assay to verify the real sites and indeed the predicted site is the most possible functional site in regulation of APP.To further verify the role of miR-16 in regulation of endogenous APP in the hippocampus of SAM mice, firstly, the in situ hybridization technology and immunohistochemical analyses have been chosen to detect the expression of miR-16 and APP in the hippocampus of SAM mice. The results showed that APP protein and miR-16 distribution in the hippocampus are complementary. miR-16 has strong expression in hippocampal pyramidal cell layer and granule cell layer, while the APP protein express intensely in other part of hippocampus. Then the stereo-positioner of brain is used to inject the miR-16 mimics and control into the lateral ventricle. The result of Western Blot shows that the expression level of APP in the mimics group was significantly lower than in the control group, but the result of RT-PCR shows that the mRNA level has no obvious change.To testify the relationship between miR-16 and APP in embryonic neuronal development of normal mouse, we also used the in situ hybridization technology and immunohistochemical analyses to profile their expressional patterns in tissue sections of embryonic 12.5,16.5. miR-16 is mainly expressed at ventricular zone,composed by neural progenitors, while APP at mature neurons. Apparently, miR-16 is complement to the expression pattern of APP.Taken together, these data revealed the abnormal lower expression of miR-16 in the AD animal model, SAMP8, compared to SAMR1, attenuates the inhibition of its target gene APP at post transcriptional level which lead to the accumulation and enrichment of APP protein in AD mouse and might be an explanation for the functional loss of both study and memory.