Inducible expression of Tau repeat domain in cell models of tauopathy: aggregation is toxic to cells but can be reversed by inhibitor drugs.
We generated several cell models of tauopathy in order to study the mechanisms of neurodegeneration in diseases involving abnormal changes of tau protein. N2a neuroblastoma cell lines were created that inducibly express different variants of the repeat domain of tau (tau(RD)) when exposed to doxycycline (Tet-On system). The following three constructs were chosen: (i) the repeat domain of tau that coincides with the core of Alzheimer paired helical filaments; (ii) the repeat domain with the deletion mutation DeltaK280 known from frontotemporal dementia and highly prone to spontaneous aggregation; and (iii) the repeat domain with DeltaK280 and two proline point mutations that inhibit aggregation. The comparison of wild-type, pro-aggregation, and anti-aggregation mutants shows the following. (a) Aggregation of tau(RD) is toxic to cells. (b) The degree of aggregation and toxicity depends on the propensity for beta-structure. (c) Soluble mutants of tau(RD) that cannot aggregate are not toxic. (d) Aggregation is preceded by fragmentation. (e) Fragmentation of tau(RD) in cells is initially due to a thrombin-like protease activity. (f) Phosphorylation of tau(RD) (at KXGS motifs) precedes aggregation but is not correlated with the degree of aggregation. (g) Aggregates of tau(RD) disappear when the expression is silenced, showing that aggregation is reversible. (h) Aggregation can be prevented by drugs and even pre-formed aggregates can be dissolved again by drugs. Thus, the cell models open up new insights into the relationship between the structure, expression, phosphorylation, aggregation, and toxicity of tau(RD) that can be used to test current hypotheses on tauopathy and to develop drugs that prevent the aggregation and degeneration of cells.
Khlistunova
Inna
I
Max-Planck-Unit for Structural Molecular Biology, DESY, Hamburg, Germany.
Biernat
Jacek
J
Wang
Yipeng
Y
Pickhardt
Marcus
M
von Bergen
Martin
M
Gazova
Zuzana
Z
Mandelkow
Eckhard
E
Mandelkow
Eva-Maria
EM
D000544:Alzheimer Disease / Q000378:metabolism; D020816:Amino Acid Motifs; D052160:Benzothiazoles; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006801:Humans; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008956:Models, Chemical; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects*; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D024801:Tauopathies / Q000473:pathology*; D013844:Thiazoles / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors; D016875:tau Proteins / Q000737:chemistry*
D052160:Benzothiazoles; D020033:Protein Isoforms; D013844:Thiazoles; D016875:tau Proteins; C009462:thioflavin T; D010447:Peptide Hydrolases; D013917:Thrombin