In vitro modelling of tau phosphorylating kinases : emphasis of Cdk5
Author: Jämsä, Anne
Date: 2007-12-18
Location: Hörsalen, Novum, plan 4, Karolinska Universitetssjukhuset, Huddinge
Time: 09.00
Department: Institutionen för neurobiologi, vårdvetenskap och samhälle / Department of Neurobiology, Care Sciences and Society
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thesis.pdf (1.179Mb)
Abstract
The main hallmarks of Alzheimer’s disease (AD) are extracellular deposits
of betaamyloid (Aβ) 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 3β (Gsk3β) contributed
most to tau phosphorylation whereas Cdk5 made a minor contribution.
Lithium, a GSK3β-inhibitor, reproducibly inhibited tau phosphorylation in
a wide concentration range indicating that this model can be used to
screen for GSK3β 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 Aβ treated hippocampal organotypic
cultures revealed increased tau phosphorylation at the Ser396 epitope
probably through activation of Gsk3β 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.
List of papers:
I. Jämsä A, Hasslund K, Cowburn RF, Bäckström A, Vasänge M. (2004). "The retinoic acid and brain-derived neurotrophic factor differentiated SH-SY5Y cell line as a model for Alzheimer's disease-like tau phosphorylation." Biochem Biophys Res Commun 319(3): 993-1000
Pubmed
II. Jämsä A, Bäckström A, Gustafsson E, Dehvari N, Hiller G, Cowburn RF, Vasänge M. (2006). "Glutamate treatment and p25 transfection increase Cdk5 mediated tau phosphorylation in SH-SY5Y cells." Biochem Biophys Res Commun 345(1): 324-31
Pubmed
III. Johansson S, Jämsä A, Vasänge M, Winblad B, Luthman J, Cowburn RF. (2006). "Increased tau phosphorylation at the Ser396 epitope after amyloid beta-exposure in organotypic cultures." Neuroreport 17(9): 907-11
Pubmed
IV. Jämsä A, Agerman K, Radesäter A-C, Ottervald J, Malmström J, Hiller G, Liu G, Vasänge M (1970). "Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening" (Manuscript)
I. Jämsä A, Hasslund K, Cowburn RF, Bäckström A, Vasänge M. (2004). "The retinoic acid and brain-derived neurotrophic factor differentiated SH-SY5Y cell line as a model for Alzheimer's disease-like tau phosphorylation." Biochem Biophys Res Commun 319(3): 993-1000
Pubmed
II. Jämsä A, Bäckström A, Gustafsson E, Dehvari N, Hiller G, Cowburn RF, Vasänge M. (2006). "Glutamate treatment and p25 transfection increase Cdk5 mediated tau phosphorylation in SH-SY5Y cells." Biochem Biophys Res Commun 345(1): 324-31
Pubmed
III. Johansson S, Jämsä A, Vasänge M, Winblad B, Luthman J, Cowburn RF. (2006). "Increased tau phosphorylation at the Ser396 epitope after amyloid beta-exposure in organotypic cultures." Neuroreport 17(9): 907-11
Pubmed
IV. Jämsä A, Agerman K, Radesäter A-C, Ottervald J, Malmström J, Hiller G, Liu G, Vasänge M (1970). "Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening" (Manuscript)
Issue date: 2007-11-27
Rights:
Publication year: 2007
ISBN: 978-91-7357-400-6
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