Physiological roles of miR-29

SUMMARY micro-RNAs are short non-coding RNA molecules that have emerged as major regulators of gene expression. One of the most studied micro-RNAs is miR-29. miR-29 encompasses a family of three micro-RNAs, encoded by two different clusters, the miR-29a/b-1 and miR-29b-2/c clusters. All miR-29 isoforms are depicted to target the same mRNAs. miR-29 is implicated in a variety of important functions in many systems and any alteration in its expression will result in perturbed functions, leading to pathologic conditions. miR-29 has been involved in functions of connective tissues, in the immune and cardiovascular systems and many roles are postulated at the level of nervous system. In this work, we investigated extensively the expression pattern of miR-29 family members in development and aging and further characterized the cell types that express miR-29. In order to validate the postulated target mRNAs under physiological conditions and explore the physiological roles of miR-29, primarily in the nervous system, we generated miR-29a/b-1 and miR-29b-2/c knockout mouse models. miR-29a/b-1 knockout models have reduced lifespan, which is further shortened in the miR-29b-2/c heterozygote background, indicating a gene-dosage dependent effect. Furthermore, the double knockout of both miR-29 clusters is extremely difficult to obtain, further supporting the notion that both miR-29 clusters act redundantly. In this thesis we analysed the more subtle, yet highly interesting effects upon deletion of the miR-29a/b-1 cluster alone. We have previously shown that miR-29 expression was altered in a subset of Alzheimer´s disease patients where BACE-1 was upregulated. Therefore, we explored the in vivo role of miR-29a/b-1 in memory and Alzheimer´s disease. Our behavioural data suggested that miR-29a/b-1 leads to memory deficits especially at older stages, concluding a role of miR-29a/b-1 in proper memory function. We further showed that miR-29a/b-1 knockout results in a modest upregulation of BACE-1 mRNA levels, but failed to show a concomitant protein level change. Possibly, the effect is so moderate because BACE-1 is still repressed by the miR-29 b-2/c isoforms. We showed that miR-29a/b-1 knockout animals have severe ataxia, which is in agreement with the observation that miR-29 is highly expressed in Purkinje cells. The ataxic phenotype is not due to Purkinje cell loss, but instead is attributed to malfunctions of cerebellar circuits. We reported that miR-29a/b-1 knockout Purkinje cells are atrophic, have a defective dendritic tree and show a reduced number of glutamatergic synapses between the parallel fiberand Purkinje cells. We further used a bioinformatics approach in an attempt to identify the targets of miR-29 and the pathways with defective function that will explain the phenotype and verified them experimentally. We identified the voltage-gated potassium channel KCNC3 as a relevant target of miR-29, but cannot exclude that the ataxic phenotype is caused by dysregulation of a more complex set of proteins. Finally we, in collaboration with Dr. Adrian Liston, identified a role of miR-29a in the immune system. miR-29a, but not miR-29b is expressed in the thymus. miR-29a is essential for proper thymic function, as miR-29a/b-1 knockout mice showed premature thymic involution. We showed that thymic involution is due to increased susceptibility of thymic epithelial cells to pathogenic signals. We further identified the pathway by which thymic epithelium suppresses thymic function and size by acting through the interferon alpha receptor pathway, which is upregulated in our miR-29a/b-1 knockout model. In essence, we propose three distinct physiological roles of miR-29, two of which involve the nervous and one the immune systems. Firstly, there is evidence for a role of miR-29 in memory impairment. Secondly, we provide evidence for miR-29 involvement in cerebellar functions, which keep balance and coordination intact. Thirdly, we present data of a miR-29 role in fine-tuning thymic output and sensitivity to baseline pathogenic signals.status: publishe