Characterizing Mir-34a Expression Patterns And Its Functions In The Brain

MicroRNAs (miRNAs) are abundant in the brain and have been implicated in neurogenesis and brain disorders. However, the essential rules of miRNAs in regulation of neuronal aging and progressive neurodegeneration have not been established. In this study, the spatial and temporal pattern of mir-34a expression in the brain was determined. In addition, several novel targets of mir-34a were validated and induction of mir-34a mediates neuronal apoptosis. Importantly, the deregulation of mir-34a expression in neurons was found to correlate with Alzheimer's pathogenesis. Moreover, the correlations between the changes of mir-34a expression in brain tumors and its targets demonstrated that miR-34a might play critical roles in brain cancer development.Part I. Characterization of miR-34a transcripts in the brain. In this part of study, we demonstrated that miR-34a expresses with aging in the brain of rhesus monkey using microarray, northern blotting and ISH. Correspondingly, the same trend was found in the primary cultures of rat cortical neurons. Furthermore, high level expression of miR-34a was found in the external granular layer and outer pyramidal layer (II/III layers). Co-localization of miRNA positive cells with a specific cellular marker Map-2 showed that mir-34a signals were exclusively detected in cell bodies of neurons. The determination of the spatial and temporal patterns of mir-34a expression in the brain provided insight into its biological functions.Part II. Prediction of mir-34a targets. The putative conserved binding sites were predicted by KeyTar, which is a sequence and structure based algorithm developed by our team. This algorithm would largely improve the accuracy of miRNA target prediction when the real status of both miRNAs and their targets in the biological systems are evaluated (unpublished data). Considering the possible rules of mir-34a and its expression patterns in the physiological and pathological conditions, the genes Bcl-6, FoxP1, Notch1, R-Ras and CCND1, are selected as the putative targets of mir-34a.Part III. Induction of mir-34a mediates neuronal apoptosis. In this part, we extended our study to test the function of this miRNA in the primary culture of rat cortical neurons. We found induction of mir-34a led to neuronal apoptosis. The data was further confirmed by the analysis of cleaved activated caspase-3. Moreover, we found that induction of miR-34a might be associated with the putative targets BCL-6. These results indicate that miR-34a mediates neuronal death versus suppressing a transcriptional repressor bcl6 that is known to direct a negative feedback loop to p53. Thus, we hypothesize that it is possible mir-34a has a critical role in neuronal development and aging.Part IV. Deregulation mir-34a is correlated with Alzheimer's pathogenesis though modulating multiple cell cycle associated factors. In this part of the study, we showed mir-34a was deregulated in the brain of a mouse AD model and in the brain of human AD. Moreover, we found FoxP1 and Notch1 are the targets of mir-34a. Correspondingly, the decreased level of mir-34a in AD neurons was detected to correlate with the increased levels of its targets Foxp1 and notch1. Of noted is that deregulations of Notch1, CCND1 and CDC25a have been known to associate with Alzheimer's pathogenesis in human. Our data demonstrate that FoxP1 is involved in Alzheimer's pathogenesis and the deregulation mir-34a is correlated with Alzheimer's pathogenesis versus modulating multiple anti-apoptotic factors. Thus, this study provides valuable clues to unravel the complex interrelationships between miRNA transcripts and neurodegenerative pathogenesis.Part V. mir-34a could promote apoptosis in brain tumor cells through translational repression of genes including R-Ras. It has been reported that mir-34a modulates the gene expression program initiated by p53 and serves as a potential tumor suppressor in a variety of tumor cells recently. In this part of the study, the spatial and temporal patterns of mir-34a expression in different species were determined. Northern Blotting and ISH analyses showed miR-34a was poorly expressed in glioma and neuroblastoma cells. Moreover, induction of mir-34a was found to promote tumor cell apoptosis. We demonstrated here firstly that miR-34a regulates R-ras pathway in brain tumors. The molecule, mir-34a, therefore, might be a possible target that could be employed in anti-brain cancer therapy.In summary, this doctoral thesis is focused on understanding molecular mechanisms how mir-34a is involved in aging and in tumor in the brain. The dissertation provides three clear contributions in this area: age-related mir-34a regulates neuron senescence, deregulation mir-34a is correlated with Alzheimer's pathogenesis and miR-34a might suppress the R-Ras pathway, thus might be a possible target that could be employed in anti-brain cancer therapy.