Cell lines expressing tau protein

Abstract

The present invention provides a cell model for studying Tau protein-related diseases.

Description

[0001] This invention provides a cell line expressing a specific Tau protein.

[0002] Tau protein aggregates into filaments or inclusion bodies in several neurodegenerative diseases, including Alzheimer's disease. Reliable model systems are needed to investigate this pathological marker and potential treatments. Developing such models has been extremely difficult due to the incomplete understanding of the pathogenesis of Tau aggregation, and cell-based models available to date in this field have shown significant limitations.

[0003] Most cell lines use aggregation inducers, such as exogenous Tau. However, exogenous Tau-induced intracellular Tau accumulation is not an empirically validated characteristic of disease pathophysiology. Furthermore, it requires additional steps, making this model less attractive for high-throughput (or even higher-throughput) research methods.

[0004] There is a need for a cellular Tau model that overcomes at least some of the shortcomings of known cellular models. Summary of the Invention

[0005] In a first aspect, the present invention provides a cell line comprising a transgene encoding 4RDΔK Tau protein (referred to as H4-Tau4RDΔK) or a transgene encoding 1N4R P301L / S320F (referred to as H4-Tau1N4R-DM) Tau protein, wherein Tau protein expression is inducible.

[0006] In embodiments of the present invention, the cell line is a stable cell line.

[0007] In an embodiment of the present invention, the cell line is the human H4 epithelial cell line.

[0008] In embodiments of the present invention, transgenic expression is controlled by the Tet promoter.

[0009] In an embodiment of the present invention, the transgene encodes the 4RDΔK Tau protein.

[0010] In an embodiment of the present invention, the transgene encodes the 1N4R P301L / S320F Tau protein.

[0011] In embodiments of the present invention, the Tau protein is human Tau protein.

[0012] In an embodiment of the present invention, the transgene encoding the 4RDΔK Tau protein has the nucleotide sequence shown in Seq. Id. No. 2. The 4RDΔK Tau protein of the present invention has the amino acid sequence shown in Seq. Id. No. 4.

[0013] In an embodiment of the present invention, the transgene encoding the 1N4R P301L / S320F Tau protein has the nucleotide sequence shown in Seq. Id. No. 1. The 1N4R P301L / S320F Tau protein of the present invention has the amino acid sequence shown in Seq. Id. No. 3.

[0014] In a second aspect, the present invention provides the use of the cell lines of the present invention in a method for identifying molecules that regulate the expression and aggregation of Tau protein.

[0015] In embodiments of the present invention, the molecules that regulate Tau protein expression and aggregation are Tau protein aggregation inhibitors or regulators. Attached Figure Description

[0016] Figure 1: H4 T-REx cells overexpressing 4RDΔK Tau (molecular weight ~14 kDa) (H4-Tau4RDΔK). Tau expression was observed from day 1 after doxycycline induction and appeared to persist until day 5 of induction (tested up to day 5). Tau expression appeared to saturate after 24 hours. T-REx cells expressed tetracycline repressor protein, thereby inhibiting Tau protein expression in the absence of doxycycline.

[0017] Figure 2: H4 T-REx cells (molecular weight ~58-60 kDa) overexpressing the 1N4R Tau isoform with P301L and S320F mutations (H4-Tau1N4R-DM). Tau expression was observed from day 1 after doxycycline induction and appeared to persist until day 5 of induction (tested up to day 5). Tau expression appeared to saturate after 24 hours. T-REx cells expressed tetracycline repressor protein, thereby inhibiting Tau protein expression in the absence of doxycycline. Abbreviation: PTM Tau - Post-translational modified Tau

[0018] Figure 3: Sodium dodecyl sarcosinate extraction separates insoluble Tau (expected to contain large aggregates and filaments) from soluble Tau (monomers and small oligomers). H4-Tau4RDΔK cells begin to form sodium dodecyl sarcosinate insoluble Tau aggregates from day 1, which remain stable until day 5 of induction (tested for 5 days).

[0019] Figure 4: The Tau preparation extracted from sodium lauryl sarcosinate separates insoluble Tau (expected to contain large aggregates and filaments) from soluble Tau (monomers and small oligomers). H4-Tau1N4R-DM cells begin to form sodium lauryl sarcosinate-insoluble Tau aggregates from day 1, with a time-dependent increase up to day 5 (tested for 5 days).

[0020] Figure 5: Tau protein aggregation as measured by HTRF-based aggregation assay in the H4-Tau1N4R-DM cell line. Similar to the sodium dodecyl sarcosinate preparation method, a time-dependent increase in Tau aggregation was observed. However, the aggregate signal only appeared on day 2. n = 3 technical replicates.

[0021] definition:

[0022] As used herein, the term "4RDΔK tau" protein refers to a Tau protein containing only the four repeating domains of the Tau protein and lacking the lysine (K) residue at position 280. The 4RDΔK Tau protein of this invention is encoded by the nucleic acid sequence shown in Seq. Id. No. 2. Abbreviation: TauRDΔK. The term "H4-Tau4RDΔK" is used for cell lines overexpressing 4RDΔK tau.

[0023] As used herein, the term "1N4R P301L / S320F tau" refers to a Tau protein containing the 1N4R Tau isoform (one N-terminal domain and four repeating domains) carrying mutant P301L and S320F. The 1N4R P301L / S320F Tau protein of this invention is encoded by the nucleic acid sequence shown in Seq. Id. No. 1. Abbreviation: Tau1N4R-DM (double mutant). The term "H4-Tau1N4R-DM" is used for cell lines overexpressing 1N4R P301L / S320F Tau.

[0024] The Tau protein isotypes with GenBank accession numbers P10636-7 (Tau E, 1N4R) and P10636-8 (Tau F, 2N4R) are naturally occurring Tau protein isotypes used to generate the Tau proteins 4RDΔK tau and 1N4R P301L / S320F Tau of the present invention. Detailed Implementation

[0025] 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 4RD (repetitive domain) Tau with K280 deletion in H4 cells (an epithelial cell line isolated from a 37-year-old male with glioma) (further referred to as Tau-4RDΔK).

[0026] By using an inducible stable cell line, we were able to control the overexpression of Tau, thereby avoiding the toxic effects of persistent Tau pathological burden on cells.

[0027] Both Tau-1N4R-DM and Tau-4RDΔK cells exhibited strong Tau aggregation upon Tau overexpression, starting from day 1 of doxycycline induction. The aggregates were detected both by Western blotting in the sodium lauryl sarcosinate insoluble fraction and, for Tau-1N4R-DM, additionally by HTR fluorescence assay. Therefore, these cell lines provide a cell-based model system with spontaneous aggregation occurring upon inducible overexpression of a mutant Tau variant without the addition of exogenous Tau. This, along with the rapid scaling-up and ease of handling of H4 cells, makes this cell model a promising tool for future research methods, including drug screening.

[0028] Example:

[0029] Cell line characterization of Tau overexpression and Tau pathology

[0030] Subculture was performed 1 to 2 times per week, at a ratio of 1:10 to 1:20.

[0031] Doxycycline-induced

[0032] Add doxycycline (#D9891) to the cell culture medium to a final concentration of 1 ug / ml.

[0033] Cell harvest

[0034] Collect the culture medium containing floating cells into 5 ml Eppendorf tubes.

[0035] Wash with 1 ml of PBS, and collect the PBS in the same tube.

[0036] Add 1 ml of accutase and incubate at 37°C for ~5 min. Collect the detached cells into the same tube (wash the wells with a portion of the culture medium removed from the tube to collect all the material).

[0037] Centrifuge and precipitate (300 xg, 5 min)

[0038] Remove supernatant

[0039] Resuspend the precipitate in ice-cold PBS and transfer it to a 1.5 ml eppendorf tube.

[0040] Centrifuge the sediment (300 xg, 5 min, 4°C, if possible).

[0041] Remove supernatant

[0042] Quickly freeze on dry ice

[0043] Store at -80℃

[0044] Cell lysis

[0045] Extraction buffer

[0046] Prepared and stored in the refrigerator:

[0047] RIPA (25 mM TrisHCl, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS)

[0048] +1 mM EGTA

[0049] +5% glycerin

[0050] Added on the day of the experiment:

[0051] +10 mM MgCl2

[0052] +2.5 mM DTT

[0053] +1x Roche PhosSTOP Tablets (PHOSS-RO)

[0054] +1x complete-minimix (Roche) EDTA-free (Roche #1836153)

[0055] +1 / 100 Recombinant DNase 1 (Roche #04536282001)

[0056] For a single confluence of 6 wells, resuspend the cell pellet in 150 μL of lysis buffer to obtain a total protein concentration of ~2–4 μg / μL (or ~100 μL of lysis buffer per 1 Mio cells).

[0057] Incubate on ice for 30 minutes

[0058] Centrifuge for 5 min, 13,000 x rpm = 15,000 x g (Heraeus Fresco microcentrifuge)

[0059] Transfer the supernatant to a new tube

[0060] Perform BCA for protein quantification (using the Pierce™ BCA Protein Assay Kit - Reducing Agent Compatible when using DTT in lysis buffer).

[0061] Sodium dodecyl sarcosinate extraction

[0062] 100 µl 15,000 x g supernatant

[0063] + Final concentration 1% Sarcosyl

[0064] Incubate at 37°C for 1.5 hours; shake at 500 RPM.

[0065] Centrifuge at 100,000 x g (4 °C) for 45 min.

[0066] Collect the supernatant (soluble fraction of sodium dodecyl sarcosinate).

[0067] (Centrifuge for 2 min, 15,000 xg)

[0068] Carefully remove all remaining supernatant.

[0069] Resuspend the precipitate in 15 µl PBS (sodium dodecyl sarcosinate insoluble fraction) + 1x PhosSTOP (PHOSS-RO)

[0070] + 1x PI cOmplete mini mix (Roche #1836153))

[0071] SDS-PAGE

[0072] Load the sample onto NuPAGE® Novex® 8% Bis-Tris MidiGel (H4-Tau1N4R-DM cell line) using MOPS run buffer or 4-12% Bis-Tris MidiGel (H4-Tau4RDΔK cell line) using MES buffer, run at 80 V for 10 min (to collect the sample), and then run at 160 V.

[0073] Sample preparation (10 μg sample per lane): Sample + 10x LDS reducing agent (Invitrogen #NP0009) + 4x LDS sample buffer (Invitrogen #NP007), add to the required volume with ddH2O.

[0074] For sodium dodecyl sarcosinate extraction:

[0075]

[0076] Boil at 95℃ for 5 minutes

[0077] Centrifuge at 13,000 rpm = 15,000 xg for 20 min (Heraeus Fresco microcentrifuge)

[0078] Pre-stained protein molecular weight standard SeeBlue Plus2 (#): 15 μL per well

[0079] Tau molecular weight standard (T7951#): Dilute 1:20 in 1x sample buffer (from 10x reducing agent and 4x LDS sample buffer) and load the sample.

[0080] Protein blot

[0081] Transfer to nitrocellulose membrane using the Trans-Blot semi-dry transfer system (Bio-Rad #170-3940): Transfer program: 25 V 7 min

[0082] Or i-Blot2 system (Invitrogen #IB23001): Transfer program: 20 V 1 min, 23 V 4 min, 25 V 2 min

[0083] Blocked in 5% Sure Block / TBSt (LubioSciences #SB232010) for 30 min.

[0084] Primary antibody: Incubate overnight at 4°C in 1% SureBlock / TBSt.

[0085] K9JA was used as the primary antibody for Tau detection.

[0086]

[0087] Secondary antibody

[0088]

[0089] Wash 3 times in 1xTBSt, 15 min each time.

[0090] Secondary antibody: Incubate at RT℃ in 1% SureBlock / TBSt for 1 h.

[0091] Wash 3 times in 1xTBSt, 15 min each time.

[0092] Detection: Incubate at RT with a 1:1 mixture of SuperSignal West Dura Extended Duration Substrate (ThermoScientific #34076) for 5 min.

[0093] Imaging using Fusion Fx (Vilber Lourmat)

[0094] Wash with TBSt three times, 15 minutes each time.

[0095] Incubate with actin HRP-conjugated AB in TBSt for 20 min.

[0096] Wash with TBSt three times, 15 minutes each time.

[0097] Detection and imaging were performed using the methods described above.

[0098] Tau aggregation HTRF determination

[0099] Dilute the sample in the kit diluent (e.g., dilute 1:10 based on the total protein concentration, e.g., dilute 1:10 for 1 μg / µl of total protein).

[0100] Prepare the antibody master mix consisting of a 1:100 dilution of anti-Tau-d2 and anti-Tau-Tb conjugates.

[0101] Take 10 μL of each sample and each control and add it to a white low-volume 384-well plate.

[0102] Add 10 μL of antibody master mixture on top.

[0103] Seal with aluminum foil and incubate at RT (4h - overnight).

[0104] Measure the emitted light at 665 nm and 620 nm.

[0105] Calculate the ratio * 10000

[0106] Normalized to the total protein concentration of each sample ( / concentration*100).

[0107] Cell culture:

[0108]

Claims

1. A cell line comprising a transgene encoding 4RDΔK Tau protein or a transgene encoding 1N4R P301L / S320F Tau protein, wherein Tau protein expression is inducible.

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

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

4. The cell line according to claims 1 to 3, wherein the transgene expression is controlled by the Tet promoter.

5. The cell line according to claims 1 to 4, wherein the transgene encodes the 4RDΔK Tau protein.

6. The cell line according to claims 1 to 4, wherein the transgene encodes the 1N4R P301L / S320FTau protein.

7. The cell line according to claims 1 to 6, wherein the Tau protein is human Tau protein.

8. The cell line according to claims 1 to 7, wherein the transgene encoding the 4RDΔK Tau protein has the nucleotide sequence shown in Seq. Id. No.

1.

9. The cell line according to claims 1 to 7, wherein the transgene encoding the 1N4R P301L / S320F Tau protein has the nucleotide sequence shown in Seq. Id. No.

2.

10. Use of the cell lines according to claims 1 to 9 in a method for identifying molecules that regulate the expression and aggregation of Tau protein.

11. The use according to claim 10, wherein the molecule regulating Tau protein expression and aggregation is a Tau protein aggregation inhibitor or regulator.

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