Alzheimer's disease (AD) is one of the most common types of senile dementia, characterized by intracellular neurofibrillary tangles (NFTs), composed of hyperphosphorylated Tau, and extracellular senile plaques, formed by the fibrillary β-amyloid (Aβ). Different kinases have been implicated in the pathogenic processes associated with AD. In particular, the CDK5 kinase has been suggested to have a prime role in the pathogenesis of AD by hyperphophorilating Tau, by mediating the neurotoxic effects triggered by Aβ, and by contributing to Aβ production. CDK5 requires for its activity the interaction with the regulatory subunit p35 or with its proteolytic product p25. Noteworthy, overexpression of p25 in transgenic mice results in increased CDK5 activity and hyperphosphorylation of Tau. p35 is encoded by CDK5R1 (Cyclin-Dependent Kinase 5 Regulatory subunit 1), a gene with a very large 3'UTR which contains several microRNA (miRNA) predicted target sites. MiRNAs are small regulatory RNAs with roles in neurodevelopment, neuroplasticity, apoptosis, and other fundamental neurobiological processes. A number of recent papers raise the possibility that misregulation of miRNAs could have a role in AD, by altering the expression of key proteins, such as BACE1 and APP. Interestingly, some of these miRNAs (miR-107, miR-15a) have many predicted target sites within CDK5R1 3'UTR.
It is currently unknown if p25/p35 levels are regulated by miRNAs and if these mechanisms are misregulated in AD, leading to an abnormal CDK5 phosphorylation activity, with an implication in AD pathogenesis. We first evidenced an inverse correlation between p35 and miR-107/103 levels in different cell lines. A significant reduction of CDK5R1 mRNA and p35 levels was observed after transfection of SK-N-BE neuroblastoma cells with the miR-103 or miR-107 precursor (pre-miR-103 or pre-miR-107). Conversely, p35 levels significantly increased following transfection of the corresponding antagonists (anti-miR-103 or anti-miR-107). Furthermore, we demonstrated, by means of luciferase assays, that miR-103 and miR-107 are able to directly interact with the CDK5R1 3'UTR, in correspondence of a specific target site. These data indicate that miR-107 and miR-103 are able to regulate p35 expression and allow us to hypothesize that a miRNA-mediated mechanism may influence CDK5 activity and the associated molecular pathways.
The present project is aimed at: to elucidate the mechanisms by which miR-107/103 and other miRNAs can regulate the expression of p35; to measure the miRNA, CDK5R1 and p25/p35 levels, the CDK5 phosphorylation activity and the Tau phosphorylation, in AD and control brain tissues, in order to evaluate if there is an inverse correlation between the levels of miRNAs and the levels of p25/p35 and CDK5 activity on Tau; to explore the possibility of a post-transcriptional modulation of CDK5 activity by transfecting miRNAs/anti-miRNAs in suitable cellular systems. The understanding of how CDK5 activity is affected by miRNAs through p35 regulation, besides providing new insights on the molecular mechanisms of AD, might allow the identification of new pharmacological targets to be used as a starting point for the setting up of new therapeutic strategies for the cure of AD.