In vitro modelling of tau phosphorylating kinases : emphasis of Cdk5
Year: 2007
Type: dissertation
Abstract: The main hallmarks of Alzheimer’s disease (AD) are extracellular deposits\nof betaamyloid (Aβ) and intracellular neurofibrillary tangles (NFT)\ncomposed of highly phosphorylated tau protein. Abnormal\nhyperphosphorylation of tau is the most deleterious step in NFT formation\nmaking the use of kinase inhibitors an attractive treatment possibility\nin AD. To enable development and screening of selective kinase\ninhibitors, well-characterized cellular assays are essential. In this\nthesis, different cell culture systems were investigated as in vitro\nmodels for tau phosphorylating kinases, with an emphasis on\ncyclin-dependent kinase-5 (Cdk5).\n\nIn paper I, differentiated SH-SY5Y cell line was investigated as a model\nfor tau phosphorylation. Sequential differentiation of SH-SY5Y cells with\nretinoic acid and brainderived neurotrophic factor induced a prominent\nincrease in the content and the phosphorylation state of tau. Of the\ninvestigated kinases, glycogen synthase kinase 3β (Gsk3β) contributed\nmost to tau phosphorylation whereas Cdk5 made a minor contribution.\nLithium, a GSK3β-inhibitor, reproducibly inhibited tau phosphorylation in\na wide concentration range indicating that this model can be used to\nscreen for GSK3β inhibitors.\n\nIn paper II, differentiated SH-SY5Y cells were treated with neurotoxic\nstimuli or transfected with p25 in order to activate Cdk5. Glutamate\nincreased Cdk5 and p35 protein levels thereby elevating Cdk5 activity and\ntau phosphorylation. When p25 was transfected to the cells, increased tau\nphosphorylation was achieved but could not be reduced with the Cdk5\ninhibitor Roscovitine. This is possibly through activation of ERK1/2,\nanother tau phosphorylating kinase, detected in Roscovitine treated\ncells. An additional finding of this study was degradation of p25 via\nproteasome in cells treated with Cdk5 inhibitors.\n\nIn paper III, investigation of Aβ treated hippocampal organotypic\ncultures revealed increased tau phosphorylation at the Ser396 epitope\nprobably through activation of Gsk3β whereas Cdk5 involvement was not\ndetected.\n\nIn paper IV, alternative Cdk5 substrates were looked for in Cdk5/p25\ntransfected HEK293 cells. A non-muscle myosin heavy chain, type B\n(NMHC-B), was identified as a novel Cdk5 substrate. Only Cdk5\nphosphorylates NMHC-B in HEK293 cells and its phosphorylation was\nconcentration-dependently inhibited with Cdk5 inhibitors. A new screening\nsystem for Cdk5 inhibitors was established using NMHC-B phosphorylation\nas a read-out.\n\nMany kinases, some with reciprocal interactions, are involved in tau\nphosphorylation in differentiated SH-SY5Y cells complicating its use as a\nmodel for Cdk5 mediated tau phosphorylation. The assay with NMHC-B\nphosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells is,\nhowever, very specific and sensitive and allows validation of compounds\ndesigned to inhibit Cdk5.
Source: Figshare
Author: Anne Jämsä
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1585044801