In vitro modelling of tau phosphorylating kinases : emphasis of Cdk5

Sammanfattning: The main hallmarks of Alzheimer’s disease (AD) are extracellular deposits of betaamyloid (A?) and intracellular neurofibrillary tangles (NFT) composed of highly phosphorylated tau protein. Abnormal hyperphosphorylation of tau is the most deleterious step in NFT formation making the use of kinase inhibitors an attractive treatment possibility in AD. To enable development and screening of selective kinase inhibitors, well-characterized cellular assays are essential. In this thesis, different cell culture systems were investigated as in vitro models for tau phosphorylating kinases, with an emphasis on cyclin-dependent kinase-5 (Cdk5). In paper I, differentiated SH-SY5Y cell line was investigated as a model for tau phosphorylation. Sequential differentiation of SH-SY5Y cells with retinoic acid and brainderived neurotrophic factor induced a prominent increase in the content and the phosphorylation state of tau. Of the investigated kinases, glycogen synthase kinase 3? (Gsk3?) contributed most to tau phosphorylation whereas Cdk5 made a minor contribution. Lithium, a GSK3?-inhibitor, reproducibly inhibited tau phosphorylation in a wide concentration range indicating that this model can be used to screen for GSK3? inhibitors. In paper II, differentiated SH-SY5Y cells were treated with neurotoxic stimuli or transfected with p25 in order to activate Cdk5. Glutamate increased Cdk5 and p35 protein levels thereby elevating Cdk5 activity and tau phosphorylation. When p25 was transfected to the cells, increased tau phosphorylation was achieved but could not be reduced with the Cdk5 inhibitor Roscovitine. This is possibly through activation of ERK1/2, another tau phosphorylating kinase, detected in Roscovitine treated cells. An additional finding of this study was degradation of p25 via proteasome in cells treated with Cdk5 inhibitors. In paper III, investigation of A? treated hippocampal organotypic cultures revealed increased tau phosphorylation at the Ser396 epitope probably through activation of Gsk3? whereas Cdk5 involvement was not detected. In paper IV, alternative Cdk5 substrates were looked for in Cdk5/p25 transfected HEK293 cells. A non-muscle myosin heavy chain, type B (NMHC-B), was identified as a novel Cdk5 substrate. Only Cdk5 phosphorylates NMHC-B in HEK293 cells and its phosphorylation was concentration-dependently inhibited with Cdk5 inhibitors. A new screening system for Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out. Many kinases, some with reciprocal interactions, are involved in tau phosphorylation in differentiated SH-SY5Y cells complicating its use as a model for Cdk5 mediated tau phosphorylation. The assay with NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells is, however, very specific and sensitive and allows validation of compounds designed to inhibit Cdk5.