A cell line expressing tau protein

Abstract

The present invention provides a cell model to study Tau protein related diseases.

Description

[0001] A cell line expressing Tau protein

[0002] The present invention provides cell lines expressing specific Tan proteins.

[0003] Tan protein aggregates into filaments or inclusions in several neurodegenerative diseases including Alzheimer’s disease. To study this pathological hallmark and potential therapeutic approaches, reliable model systems are needed. Since the pathomechanism of Tau aggregation is not fully understood, development of such models has been difficult and so far, the cell-based models available in the field show severe limitations.

[0004] Most cell lines use aggregation inducers such as exogenous Tau. The exogenous Tau induced intracellular Tau aggregation is not a validated representation of the pathophysiology of the disease. Furthermore, it requires additional steps, making the model less attractive for high(er) throughput research approaches.

[0005] There is a need for cellular Tau models which overcome at least some of the shortcomings of the known cellular models.

[0006] Summary of the invention:

[0007] In a first aspect, the present invention provides a cell line comprising a transgene coding for 4RDAK Tau protein (referred to as H4-Tau4RDAK) or a transgene coding for 1N4R P301L / S320F (referred to as H4-TaulN4R-DM) Tau protein, wherein the Tau protein expression is inducible.

[0008] In an embodiment of the present invention, the cell line is a stable cell line.

[0009] In an embodiment of the present invention, the cell line is a human H4 epithelial cell line.

[0010] In an embodiment of the present invention, the transgene expression is under control of a Tet promoter.

[0011] In an embodiment of the present invention, the transgene codes for 4RDAK Tau protein.

[0012] In an embodiment of the present invention, the transgene codes for 1N4R P301L / S320F Tau protein.

[0013] In an embodiment of the present invention, the Tau protein is a human Tau protein.

[0014] In an embodiment of the present invention, the transgene coding for 4RDAK Tau protein has the nucleotide sequence set forth in Seq. Id. No. 2. The 4RDAK Tau protein of the present invention has the amino acid sequence set forth in Seq. ID. No. 4.

[0015] In an embodiment of the present invention, the transgene coding for 1N4R P301L / S320F Tau protein has the nucleotide sequence set forth in Seq. Id. No. 1. The 1N4R P301L / S320F Tau protein of the present invention has the amino acid sequence set forth in Seq. ID. No. 3.

[0016] In a second aspect, the present invention provides the use of the cell lines of the present invention in a method for the identification of molecules modulating Tau protein expressions and aggregation.

[0017] In an embodiment of the present invention, the molecule modulating Tau protein expression and aggregation is a Tau protein aggregation inhibitor or modulator.

[0018] Short description of the figures:

[0019] Fig. 1: H4 T-REx cells overexpressing 4RDAK Tau (Molecular weight ~14 kDa) (H4- Tau4RDAK). Upon induction with doxycycline, Tau expression was observed starting from day 1 and appearing to stay expressed until day 5 of induction (tested until day 5). Tau expression seems to reach saturation after 24 hours. T-REx cells express the tetracycline repressor protein, repressing Tau protein expression in the absence of doxycycline. Fig. 2: H4 T-REx cells overexpressing 1N4R Tan isoform with P301L and S320F mutations (Molecular weight ~58-60kDa) (H4-TaulN4R-DM). Upon induction with doxycycline, Tau expression was observed starting from day 1 and appearing to stay expressed until day 5 of induction (tested until day 5). Tau expression seems to reach saturation after 24 hours. T-REx cells express the tetracycline repressor protein, repressing Tau protein expression in the absence of doxycycline . Abbreviation: PTM Tau - Post translationally modified Tau

[0020] Fig. 3: Sarkosyl extraction separates insoluble Tau (expected to contain bigger aggregates and filaments) from soluble Tau (monomers and small oligomers). H4-Tau4RDAK cells form sarkosyl insoluble Tau aggregates starting on day 1, stable until 5 days of induction (5 days tested).

[0021] Fig. 4: Sarkosyl extraction Tau preparation separates insoluble Tau (expected to contain bigger aggregates and filaments) from soluble Tau (monomers and small oligomers). H4-TaulN4R-DM cells form sarkosyl insoluble Tau aggregates starting on day 1, with a time-dependent increase until day 5 (5 days tested)

[0022] Fig. 5: Tau protein aggregation measured by HTRF based aggregation assay for H4-TaulN4R- DM cell line.. Similar to the sarkosyl preparation method, time dependent increase in the Tau aggregation is observed. However, the signal for aggregates appears only at day 2. n = 3 technical replicates.

[0023] Definitions:

[0024] The term “4RDAK tau” protein as used herein refers to a Tau protein comprising only the four repeat domains of the Tau protein with a deletion of lysine (K) at 280 position. The 4RDAK Tau protein of the present invention is encoded by the nucleic acid sequence set forth in Seq. Id. No. 2. In short: TauRDAK. The term “H4-Tau4RDAK” is used for the cell line overexpressing 4RDAK tau.

[0025] The term “1N4R P301L / S320F tau” protein as used herein refers to a Tau protein comprising 1N4R Tau isoform (one N-terminal domain and the four repeat domains) carrying the mutations P301L and S320F. The 1N4R P301L / S320F Tau protein of the present invention is encoded by the nucleic acid sequence set forth in Seq. Id. No. 1. In short: TaulN4R-DM (double mutant). The term “H4-TaulN4R-DM” is used for the cell line overexpressing 1N4R P301L / S320F Tau.

[0026] Tau protein isoforms with GenBank Accession No. P10636-7 (Tau E, 1N4R) and P10636-8 (Tau F, 2N4R) are the naturally occuring Tau protein isoforms used for the generation of the Tau proteins 4RDAK tau and 1N4R P301L / S320F Tau of the present invention.

[0027] Detailed description of the invention

[0028] The cell model of the present invention uses an inducible system to overexpress 1N4R P301L / S320F Tau (further referred to as Tau-1N4R-DM) or 4 RD (repeat domain) Tau with a deletion of K280 (further referred to as Tau-4RDAK) in H4 cells, an epithelial cell line isolated form a 37 year old male with neuroglioma.

[0029] By using an inducible stable cell line, we are able to control the overexpression of Tau, avoiding toxic effects of constant Tau pathology burden on the cells.

[0030] Both Tau-1N4R-DM and Tau-4RDAK cells show robust Tau aggregation upon Tau overexpression, starting from 1 day of doxycycline induction. Aggregates were detected both in sarkosyl insoluble fraction by Western blotting and for Tau-1N4R-DM additionally in an HTR fluorescence assay. Thus, these cell lines provide a cell -based model system with spontaneous aggregation upon inducible overexpression of mutant Tau variants without the need to add exogenous Tau. This and the fact that H4 cells are rapidly upscaled and easy to handle makes this cell model a promising tool for future approaches including drug screening. Examples:

[0031] Characterization of cell lines regarding Tau overexpression and Tau pathology

[0032] Subculturing 1-2 times per week 1:10 - 1:20 ratio

[0033] Doxycycline induction

[0034] • Add doxycycline (#D9891) to a final concentration of 1 ug / ml in the cell culture medium

[0035] Cell harvest

[0036] • Collect medium with floating cells in 5 ml eppendorf tube

[0037] • Wash with 1 ml PBS, collect PBS in the same tube

[0038] • Add 1 ml accutase, incubate 37 °C (~5 min), collect detached cells in same tube (wash the well with some medium from the tube to collect all material)

[0039] • Spin down (300 xg, 5 min)

[0040] • Remove supernatant

[0041] • Resuspend pellet in ice cold PBS and transfer to 1.5 ml eppendorf tube

[0042] • Spin down (300 xg, 5 min, 4 °C if possible)

[0043] • Remove supernatant

[0044] • Snap freeze on dry ice

[0045] • Store at -80 °C

[0046] Cell lysis

[0047] Extraction buffer

[0048] Prepared, stored in fridge:

[0049] RIPA (25 mM TrisHCl, 150 mM NaCl, 1 % NP40, 1 % sodium deoxycholate, 0.1 % SDS)

[0050] + 1 mM EGTA

[0051] + 5% Glycerol

[0052] Add on the day of experiment:

[0053] + 10 mM MgC12

[0054] + 2.5 mM DTT

[0055] + lx Roche PhosSTOP tablets (PHOSS-RO)

[0056] + lx complete-minimix (Roche) EDT A fr ee (Roche #1836153)

[0057] + 1 / 100 DNase 1 recombinant (Roche #04536282001)

[0058] • For 1 confluent 6 well, resuspend cell pellet in 150 ul lysis buffer to yield total protein concentrations of ~ 2-4 ug / ul (or ~ 100 ul lysis buffer per 1 Mio cells)

[0059] • Incubate on ice for 30 min

[0060] • Centrifuge 5 min 13 000 x rpm = 15 000 xg (Heraeus Fresco Microcentrifuge)

[0061] • Transfer supernatant to fresh tube

[0062] • Perform BCA for protein quantification (when using DTT in the lysis buffer, use Pierce™ BCA Protein Assay Kit - Reducing Agent Compatible)

[0063] Sarkosyl Extraction 100 jxl 15 OOOxg supernatant

[0064] • + final 1 % Sarcosyl

[0065] • 1.5h incubation at 37°C ; shaking at 500 RPM

[0066] • Centrifugation for 45 min at 100 OOOxg (4°C)

[0067] • Collect the supernatant (sarkosyl soluble fraction)

[0068] • (Centrifugation 2 min 15 000 xg

[0069] • Remove all remaining supernatant carefully)

[0070] • Resuspend the pellet in 15 pl PBS (sarkosyl insoluble fraction)

[0071] + Ix PhosSTOP (PHOSS-RO) o + lx PI cOmplete mini mix (Roche #1836153))

[0072] SDS-PAGE

[0073] Load onNuPAGE® Novex® 8 % Bis-Tris Midi Gel with MOPS running buffer (H4-TaulN4R-DM cell line) or 4-12 % Bis-Tris Midi Gel with MES buffer (H4-Tau4RDAK cell line) run 10 min 80 V (to collect sample), then at 160 V.

[0074] • Sample preparation (total 10 ug sample per lane)

[0075] Sample

[0076] + lOx LDS reducing agent (Invitrogen #NP0009)

[0077] + 4x LDS sample buffer (Invitrogen #NP007) fill with ddH2O to desired volume

[0078] For Sarkosyl extraction:

[0079] • Boil for 5 min at 95 °C

[0080] • Centrifugation for 20 min at 13 000 rpm = 15 000 xg (Heraeus Fresco Microcentrifuge)

[0081] • Protein ladder prestained SeeBlue Plus2 ( #): 15 ul per well

[0082] • Tau ladder (T7951#): 1 :20 dilution in lx sample buffer (from lOx reducing agent and 4x LDS sample buffer), load

[0083] Western Blotting

[0084] • Transfer to nitrocellulose membrane using Trans-Blot Semi-Dry Transfer system (Bio-Rad #170- 3940): Transfer protocol: 25 V 7 min

[0085] • Or i-Blot2 system (Invitrogen #IB23001): Transfer protocol: 20 V Imin, 23 V 4 min, 25 V 2min

[0086] • Blocking for 30 min in 5% Sure Block / TBSt (LubioSciences #SB232010)

[0087] • Primary antibody : overnight at 4°C in 1% SureBlock / TBSt

[0088] Use K9JA as primary antibody for Tau detection

[0089] Secondary antibody

[0090] • Wash 3x15 min in IxTBSt

[0091] • Secondary antibody : Ih at RT°C in 1% SureBlock / TBSt

[0092] • Wash 3x15 min in IxTBSt

[0093] • Detection :Incubate 5 min at RT with SuperSignal West Dura Extended Duration Substrate solution (ThermoS cientific #34076) 1:1 mixture

[0094] • Image using Fusion Fx (Vilber Lourmat)

[0095] • Wash 3x 15 min TBSt

[0096] • 20 min incubation with Actin HRP coupled AB in TBSt

[0097] • Wash 3x 15 min TBSt

[0098] • Detection and imaging as described above

[0099] Tan aggregation HTRF assay

[0100] • Dilute samples (e.g. 1:10 based on total protein concentration, e.g. 1 : 10 for 1 ug / ul total protein) in kit diluent

[0101] • Make antibody mastermix of 1 : 100 dilution of anti Tau-d2 and anti Tau-Tb conjugate

[0102] • Pipette 10 ul of each sample and each control into white low volume 384-well plate

[0103] • Add 10 ul of antibody mastermix on top

[0104] • Seal with foil and incubate at RT (4h - overnight)

[0105] • Measure emission at 665 nm and 620 nm

[0106] • Calculate ratio * 10000

[0107] • Normalize to total protein concentration of each sample ( / concentration *100)

[0108] Cell cultivation:

Claims

Claims1. A cell line comprising a trans gene coding for 4RDAK Tan protein or a trans gene coding for 1N4R P301L / S320F Tan protein, wherein the Tan protein expression is inducible.

2. The cell line of claim 1, wherein the cell line is a stable cell line.

3. The cell of claim 1 or 2, wherein the cell line is a human H4 epithelial cell line.

4. The cell line of claims 1 - 3, wherein the transgene expression is under control of a Tet promoter.

5. The cell line of claims 1 - 4, wherein the transgene codes for 4RDAK Tau protein.

6. The cell line of claims 1 - 4, wherein the transgene codes for 1N4R P301L / S320F Tau protein.

7. The cell line of claims 1 - 6, wherein the Tau protein is a human Tau protein.

8. The cell line of claims 1 - 7, wherein the transgene coding for 4RDAK Tau protein has the nucleotide sequence set forth in Seq. Id. No. 1.

9. The cell line of claims 1 -7, wherein the transgene coding for 1N4R P301L / S320F Tau protein has the nucleotide sequence set forth in Seq. Id. No. 2.

10. Use of the cell line of claims 1 - 9 in a method for the identification of molecules modulating Tau protein expressions and aggregation.

11. The use of claim 10, wherein the molecule modulating Tau protein expression and aggregation is a Tau protein aggregation inhibitor or modulator.

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