Alzheimer’s disease (AD) is characterized by the presence of neurofibrillary tangles (NFTs) made up of the Tau protein in an abnormally hyperphosphorylated state; this abnormal phosphorylation of Tau appears to be the most deleterious step in neurofibrillary degeneration. CDK5 kinase is involved in the formation of the Tau pathology in AD, hyperphophorylating Tau at different sites. CDK5 requires for its activity the interaction with p35 or p39, or with their proteolytic products p25 or p29, respectively, which are generated by digestion with calpains. It has been shown that overexpression of p25 in transgenic mice results in increased CDK5 activity and hyperphosphorylation of Tau.
The CDK5 regulatory subunit p35 is encoded by CDK5R1 (Cyclin-Dependent Kinase 5 Regulatory subunit 1). CDK5R1 displays a very conserved and long 3’UTR (2.7 Kb), a feature suggesting its involvement in the post-transcriptional control of CDK5R1 expression. We predicted in CDK5R1 3’UTR a high number of target sites miRNAs, notably miR-107. MiR-107 has been recently implicated in the acceleration of AD progression. Following over expression of miR-107 by transfection of the specific precursor in neuroblastoma SK-N-BE cells, we observed a significant reduction in p35 expression, while the transfection of anti-miR-107 led to an increase of p35 levels. These findings suggest that a deregulation of miR-107 might be implicated in AD pathogenesis by affecting CDK5R1 levels and, consequently, CDK5 phophorylation activity on Tau.
In order to identify and validate the functionality of miR-107 binding sites in CDK5R1 3’UTR, we will generate luciferase constructs with different 3’UTR portions, containing miR-107 target sites or with deleted/mutated target sites. Co-transfection of miR-107/anti-miR-107 and the 3’UTR constructs in neuronal-derived cell lines, will help to elucidate the role of each target site on transcript regulation. Similarly, we will assess whether other miRNAs may be involved in CDK5R1 regulation. A Real-Time PCR expression analysis of CDK5R1 and various miRNAs will be then performed on frozen brain tissues derived from AD patients and control individuals, to verify whether a co-deregulation of some of the analyzed miRNAs and CDK5R1 exists in AD. P25/p35 levels and CDK5 phosphorylation activity on Tau will be also measured in AD and control tissues, in order to asses if there is a correlation among the levels of the miRNAs, CDK5R1, p25/p35, CDK5 activity and Tau phosphorylation, and if these levels are altered in AD tissues. Subsequently, studies of the effects of miRNAs/anti-miRNA transfection in neurons and immortalized neuronal cell lines, especially on p25/p35 levels and CDK5 activity on Tau will be performed. Microarray and 2D-PAGE experiments on neural cells transfected with the selected miRNAs are also planned, in order to identify the genes whose expression is specifically downregulated by these miRNAs. The present study is aimed at identifying the molecular mechanisms at the basis of p25/p35/CDK5 regulation, also providing new insights into the causes of neurofibrillary degeneration in AD, with the perspective of identifying strategies that might be the starting point for the setting up of novel pharmacological therapies of Alzheimer’s disease.