SYSTEM
20230266303 · 2023-08-24
Inventors
- Charles Robert Harrington (Aberdeen, GB)
- Saskia Julie POLLACK (Brighton, GB)
- Karen Elizabeth MARSHALL (Brighton, GB)
- Louise Charlotte Serpell (Brighton, GB)
- Claude Michel Wischik (Aberdeen, GB)
Cpc classification
G01N33/5008
PHYSICS
International classification
Abstract
The present invention provides a system for the study of tau protein aggregation in neuronal cells in vitro which can be used to screen agents for therapeutic effectiveness against aggregates of tau protein or fragments thereof.
Claims
1. A method of screening for an agent effective in inhibiting cytotoxicity of a fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto to a neuronal cell comprising the steps of: (a) culturing the neuronal cell in the presence of the agent and subsequently culturing the neuronal cell with the fragment of Tau protein in the absence of heparin; or (b) culturing the neuronal cell with the fragment of Tau protein in the absence of heparin and subsequently culturing the neuronal cell in the presence of the agent; and (c) determining the cytotoxicity of the fragment of Tau protein to the neuronal cell after performing step (a) or step (b).
2. A method of screening for an agent effective in inhibiting internalisation of a fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto comprising the steps of: (a) culturing the neuronal cell in the presence of the agent and subsequently culturing the neuronal cell with the fragment of Tau protein in the absence of heparin; or; (b) culturing the neuronal cell of with the fragment of Tau protein in the absence of heparin and subsequently culturing the neuronal cell in the presence of the agent; and (c) determining the internalisation of the fragment of Tau protein in the neuronal cell after performing step (a) or step (b).
3. A method of screening for an agent effective in disrupting interaction of a fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto with endogenous tau protein comprising the steps of: (a) culturing the neuronal cell in the presence of the agent and subsequently culturing the neuronal cell with the fragment of Tau protein in the absence of heparin; or; (b) culturing the neuronal cell with the fragment of Tau protein in the absence of heparin and subsequently culturing the neuronal cell in the presence of the agent; and (c) determining the extent of interaction of the fragment of Tau protein with endogenous tau protein in the neuronal cell after performing step (a) or step (b).
4. A method as claimed in any one of claims 1 to 3, wherein the fragments of Tau protein as defined above for use according to the invention may be from 70 to 97 amino acids in length, optionally from 71 to 97 amino acids in length.
5. A method as claimed in claim 4, wherein the fragment of Tau protein is selected from the group of fragments of 71, 73, 94, 95, 97 amino acids in length.
6. A method as claimed in any one of claims 1 to 3, wherein the fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto is selected from the group consisting of fragments of Tau protein having amino acid sequences as described in SEQ ID NO:4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or a sequence with at least 85% identity thereto.
7. A system for screening for an agent effective in inhibiting cytotoxicity of a fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto comprising (i) a neuronal cell line. (ii) a fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto; and (iii) a library of agents; wherein the neuronal cell line is free from heparin.
8. A system for screening for an agent effective in inhibiting internalisation of a fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto comprising (i) a neuronal cell line. (ii) a fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto; and (iii) a library of agents; wherein the neuronal cell line is free from heparin.
9. A system for screening for an agent effective in disrupting interaction of a fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto with endogenous tau protein comprising (i) a neuronal cell line. (ii) a fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto; and (iii) a library of agents; wherein the neuronal cell line is free from heparin.
10. A system as claimed in any one of claims 7 to 9, wherein the fragments of Tau protein as defined above for use according to the invention may be from 70 to 97 amino acids in length, optionally from 71 to 97 amino acids in length.
11. A system as claimed in claim 10, wherein the fragment of Tau protein is selected from the group of fragments of 71, 73, 94, 95, 97 amino acids in length.
12. A system as claimed in any one of claims 7 to 9, wherein the fragment of Tau protein comprising at least 70 amino acids in the amino acid sequence of dGAE95 (SEQ ID NO:4) or a sequence with at least 85% identity thereto is selected from the group consisting of fragments of Tau protein having amino acid sequences as described in SEQ ID NO:4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or a sequence with at least 85% identity thereto.
Description
[0147] The present invention will now be further described with reference to the following Examples which are included for the purposes of reference only and should not be construed as being limitations on the invention, reference is also made to a number of figures in which:
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MATERIALS AND METHODS
[0158] Preparation of Recombinant dGAE
[0159] Purified recombinant truncated tau (dGAE, corresponding to amino acid residues 297-391 using numbering from 2N4R tau) was used throughout the study. Recombinant tau protein 297-391 was purified as previously described in Al-Hilaly et al (J. Mol. Biol., vol. 429, no. 23, pp 3650-3665 (2017)). Following purification, dGAE exists in a predominantly random coil conformation and consists mainly of soluble monomer and dimer as previously characterised and described in Al-Hilaly et al (J. Mol. Biol., vol. 429, no. 23, pp 3650-3665 (2017)).
Alexa Fluor® 488 Labelling of Tau Protein
[0160] To generate fluorescently tagged tau protein (dGAE-488), dGAE was covalently labelled with Alexa Fluor 488® (Life Technologies) by mixing 200 μl protein (425.2 μM) with 10 μl 113 nM Alexa Fluor® TFP ester and 20 μl 1M sodium bicarbonate (pH 8.3). The mixture was left to incubate in the dark for 15 minutes at room temperature. Zeba 7K MWCO columns (Thermo Scientific) were equilibrated by adding 1 ml 10 mM phosphate buffer (pH 7.4) and centrifuging at 1,000×g for 2 minutes at 4° C. The eluate was discarded and the process was repeated three times. The protein/dye mixture was added drop-wise onto the top of the column immediately followed by 40 μl phosphate buffer and was centrifuged at 1,000×g for 2 minutes at 4° C. The protein solution was kept on ice and the absorbance at 280 nm (A.sub.280) was measured using a NanoDrop spectrophotometer. The protein concentration was calculated using the A.sub.280, and the molar extinction coefficient of dGAE (1,400 cm.sup.−1 M.sup.−1), taking into account the absorption of the dye at A.sub.494. dGAE has 14 lysine residues, which are all potential sites at which the 488 dye can bind. The extent of labelling was determined by the A.sub.494 and the molar extinction coefficient of the dye (71,000 cm.sup.−1 M.sup.−1). The protein (dGAE-488) was used immediately for subsequent experiments or subjected to agitation in vitro.
In Vitro Assembly of dGAE and dGAE-488
[0161] In vitro assembly of dGAE and dGAE-488 was performed as previously described in Al-Hilaly et al (J. Mol. Biol., vol. 429, no. 23, pp 3650-3665 (2017)) without reducing agent. Briefly, 100 μM protein was diluted in 10 mM phosphate buffer (pH 7.4) and incubated at 37° C. whilst agitating with a speed of 700 rpm on an Eppendorf ThermoMixer® for 72 hours. Samples were visualised using negative stain transmission electron microscopy (TEM). All experiments were conducted using the stock 100 μM dGAE in phosphate buffer.
Negative Stain TEM
[0162] Aliquots (4 μl) of dGAE assembly mixtures (100 μM in phosphate buffer, pH 7.4) were placed on 400-mesh carbon-coated grid and incubated for 1 minute. After removing excess solution with filter paper, the grid was washed with 4 μl filtered Milli-Q water for 1 minute and blotted. The grids were negatively stained with 4 μl filtered 2% (w/v) uranyl acetate for 1 minute, blotted dry and left to air dry for at least five minutes. Grids were examined on a JEOL JEM1400-Plus Transmission Electron Microscope at 100 kV and electron micrograph images were collected using 4k×4k One View (Gatan) camera.
Circular Dichroism Spectroscopy
[0163] Circular dichroism spectroscopy (CD) was performed using a Jasco Spectrometer J715 and spectra collected in triplicate at a maintained temperature of 21° C. Protein samples were placed into 0.02 mm path length quartz cuvettes (Hellma) and scanned from 180 to 320 nm. CD data were converted to molar ellipticity (deg.Math.cm.sup.2.Math.dmol.sup.−1).
Cell Culture and dGAE Treatment
[0164] Undifferentiated SH-SY5Y human neuroblastoma cells were grown in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% Foetal Calf Serum (FCS), 1% Penicillin/Streptomycin (P/S) and 1% L-glutamine. For differentiation, SH-SY5Y cells were plated at a density of 50,000 cells per well in 24-well plates or 300,000 cells per well in 6-well plates. For immunofluorescence, cells were plated onto Menzel-Gläser coverslips (Thermo Scientific). For live-cell imaging, cells were plated on 35-mm dishes on a 1.5-mm coverslip (Mattek). On the first day of differentiation, media was replaced with low-serum culture media (DMEM/F-12 containing 1% FCS, 1% P/S and 1% L-glutamine) containing 10 μM all-trans retinoic acid (RA) (Sigma-Aldrich) and cells were incubated for 48 h. On day 3, this process was repeated with fresh RA and cells were incubated for a further 48 h. On day 5, the cells were washed once in serum-free culture media to remove traces of serum. Serum-free culture media (DMEM/F-12 containing 1% P/S and 1% L-glutamine) containing 50 ng/ml brain-derived neurotrophic factor (STEMCELL Technologies) was added to the cells and incubated for 48 h. Differentiated cells (dSH-SY5Y) were ready to use for experiments on day 7. For dGAE treatment, cells were exposed to 1 μM dGAE (either soluble without agitation or after agitation) and incubated for 24 h. All experiments were conducted using dSH-SY5Y cells.
Cell Viability Assay
[0165] Following the addition of dGAE, cell viability was measured using ReadyProbes® Cell Viability Imaging Kit (Life Technologies). The kit contains a NucBlue® reagent to label all cells (blue) and a NucGreen® reagent to label dead cells only (green). For tagged dGAE, NucRed® reagent was used to label dead cells (red). One drop of each reagent was added to cells in 500 μl media as described in the manufacturers protocol (Life Technologies). Cells were incubated with the reagents at 37° C. for 15 minutes and the media was replaced with Live Cell Imaging Solution (Invitrogen™, Thermo Scientific). Cells were imaged using a Zeiss Cell Observer Axiovert 200M microscope. DAPI fluorescence was captured using a G 365 excitation filter and a LP 420 emission filter with a FT 395 dichroic. Green fluorescence was captured using a FITC filter set (BP 450-490 excitation filter, BP 515-565 emission filter and FT 510 dichroic). Identical acquisition settings were used for all replicates and images were analysed using FIJI. Six fields of view were taken per sample and an average of 4500 cells were analysed per condition. The proportion of buffer-treated cell death was quantified by converting the images to grayscale followed by manually adjusting the threshold and converting it into a binary image to highlight live DAPI-stained cells. The number of cells was automatically counted. The dead cells were counted in the same way and cell death was expressed as a percentage of buffer-treated cells.
Cell Lysis and Fractionation
[0166] Cells were detached from the coverslip by incubation in 0.25% trypsin-EDTA (Gibco™) and then mixed with 5 ml culture media. The cells were harvested by centrifugation at 500×g for 5 minutes and the supernatant was discarded. The cell pellet was resuspended in ice-cold PBS and centrifuged at 500×g at 4° C. Cells were lysed in 1% Triton lysis buffer (1% Triton X-100 (v/v), 150 mM NaCl and 50 mM Tris-HCl, pH 7.6) containing Halt™ protease inhibitors (Thermo Scientific) and phosphatase inhibitors (Thermo Scientific) for 15 minutes on ice. The samples were centrifuged at 16,000×g for 30 minutes at 4° C., and the supernatant was collected (Triton-soluble fraction). The pellet was resuspended in SDS lysis buffer (1% SDS (w/v), 150 mM NaCl and 50 mM Tris-HCl, pH 7.6) containing protease and phosphatase inhibitors. The samples were centrifuged at 16,000×g for 30 minutes at room temperature and the supernatant was collected (Triton-insoluble fraction). Protein concentration in the Triton-soluble fractions were determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's instructions.
SDS-PAGE and Western Blotting
[0167] For cell lysates, Triton-soluble protein (20 μg) and an equal volume of Triton-insoluble protein were each mixed with Læmmli sample buffer (4×) (Bio-Rad Laboratories) containing 5% (v/v) p-mercaptoethanol and heated at 95° C. for 5 minutes. For recombinant protein, 3 μg was mixed with sample buffer without reducing agent or boiling. All samples were centrifuged for five minutes at 1000×g and samples were loaded onto 4-20% Mini-PROTEAN® precast gels (Bio-Rad Laboratories) and run at 120 V for 1 h in Tris-glycine running buffer (25 mM Tris, 192 mM glycine, pH 8.3), or until the sample buffer reached the end of the gel. For Coomassie staining, the gel was washed three times in double distilled water for five minutes and stained with Imperial™ protein stain (Thermo Scientific) for 1 h and then destained overnight in double distilled water. The stained gel was scanned using an HP Photosmart C5280 scanner. For western blotting, the separated proteins on the gel were transferred to nitrocellulose membrane (0.45 μm) at 200 mA for 90 minutes. The membranes were blocked in 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline (50 mM Tris-HCl, pH7.4, 150 mM NaCl) containing 0.1% (v/v) Tween-20 (TBS-T) for 1 h rocking at room temperature. Membranes were incubated with primary antibodies diluted in 5% BSA in TBS-T overnight at 4° C. The following primary antibodies and dilutions were used; Anti-Tau (polyclonal, total tau) (Thermo Scientific) (1:2500), AT180 (anti-pT231) (Thermo Scientific) (1:1000), AT8 (anti-pS202-T205) (1:1000) (Thermo Scientific), anti-GAPDH (1:5000) (Abcam). The next day, membranes were incubated in horseradish peroxidase (HRP)-anti-mouse antibody (1:5000) (Sigma-Aldrich) or HRP-anti-rabbit antibody (1:5000) (Abcam) in 5% BSA in TBS-T for 1 h at room temperature. The membranes were washed 3×10 mins in TBS-T between antibody incubations. Immunoreactive protein bands were detected using ECL substrate (Clarity™, Bio-Rad) and X-ray films were scanned using an HP Photosmart C5280 scanner. FIJI was used to quantify the bands in arbitrary densitometry units. The density of bands corresponding to full-length tau (50-70 kDa) were determined and normalised against amount of GAPDH. These values were used to calculate the proportion of total tau that is phosphorylated, expressing the proportions as a percentage of the buffer-treated control group. For the quantification of Triton-insoluble and soluble protein, the proportion of tau in the insoluble and soluble fraction was calculated using the equations insoluble/(soluble+insoluble) or soluble/(soluble+insoluble), respectively and expressed as a percentage of the respective soluble or insoluble control value.
Immunofluorescence
[0168] Cell culture medium was aspirated from dSH-SY5Y cells and washed once with PBS. Cells were fixed in 4% (w/v) paraformaldehyde (PFA) in PBS for 15 minutes followed by three washes in PBS. For permeabilization, the cells were incubated in 0.25% (v/v) Triton X-100 in PBS for 10 minutes. Cells were blocked in 2% (w/v) BSA in PBS for 1 h, followed by three washes in PBS. Primary antibodies diluted in 2% (w/v) BSA in PBS and were incubated with the cells for 1 h. The primary antibodies and dilutions used were; AT180 (anti-pT231) (1:250) (Thermo Scientific), AT8 (anti-pS202-T205) (1:500) (Thermo Scientific) and Tau1 (dephosphorylated tau at S195, 198, 199 and 202) (Merck Millipore). Cells were incubated with goat anti-mouse-Alexa Fluor® 594 (1:1000) (Invitrogen™, Thermo Scientific) diluted in 2% (w/v) BSA in PBS for 1 h in the dark. Cells were washed 3 times in PBS between antibody incubations. Cells were mounted onto glass slides using Prolong Gold mounting medium containing 4,6-diamidino-2-phenylindole (DAPI). Mounted slides were stored in the dark at room temperature for 24-48 h before imaging and kept at 4° C. for long term storage. Cells were imaged using Leica SP8 confocal microscope.
Labelling of Acidic Organelles
[0169] Cells were plated on 35-mm dishes on a 1.5-mm coverslip (Mattek). Following differentiation, dGAE-488 (5 μM) was added to the cells for 24 h. For labelling lysosomes and endosomes, LysoTracker® red (Life Technologies) was diluted in culture media at a final concentration of 50 nM and incubated for 90 mins before imaging using a Leica SP8 confocal microscope.
Confocal Microscopy
[0170] All confocal microscopy imaging was carried using a Leica SP8 confocal microscope. The instrument setting used PMT 3 and PMT Trans channels/lasers and images were acquired with a HC PLAPoCs2 63×/1.40 oil-immersion objective lens. Samples were scanned sequentially to prevent spectral bleed through. All images were collected as Z-stacks for all channels using a step size of 0.5 μm. Five to ten Z-stacks were taken for each sample and each experiment was repeated three times or more. To monitor live uptake of dGAE-488, the environment was maintained at 37° C. with humidified CO.sub.2 and the Adaptive Focus Control feature was used to maintain constant focal planes throughout the course of the experiment.
Processing Cells for TEM
[0171] DSH-SY5Y cells were treated with Alexa Fluor® 488 containing buffer, or 10 μM freshly prepared dGAE-488 for 24 h. The cells were washed once in PBS, scraped into a tube and centrifuged at 500×g for 5 minutes. The media was removed and the cells were suspended in a 1:1 mixture of pre-warmed culture media: 4% (v/v) PFA for 15 minutes at 37° C. The cells were centrifuged at 500×g for 5 mins and the cells were resuspended in fresh 4% (v/v) PFA and 0.1% (v/v) glutaraldehyde (GA) in 0.1 M phosphate buffer (pH 7.4) for 3 h at room temperature. The fixed cells were centrifuged at 1,000×g for 5 minutes. The supernatant was discarded and the pellet was suspended in 50 mM glycine in PBS for 10 minutes at room temperature. The cells were centrifuged at 1,000×g for 5 minutes and the pellet was washed three times with 0.1 M cacodylate buffer (pH 7.4). Around 200 μl 4% (w/v) low melting point agarose was added to the cells and immediately centrifuged at 1,000×g for 10 minutes at 30° C. The tube was immediately transferred to 4° C. or on ice for 20 minutes to solidify the agarose. The agarose-embedded cell pellet was transferred to a new tube and washed 2-3 times with 0.1 M cacodylate buffer (pH 7.4). The pellet was post-fixed in a reduced osmium solution (1% (v/v) osmium tetroxide, 1.5% (w/v) potassium ferrocyanide in 0.1 M cacodylate buffer, pH 7.4) for 1 h at 4° C. followed by washing three times in 0.1 M cacodylate buffer (pH 7.4) and three times in double-distilled H.sub.2O for 5 minutes each. The pellet was dehydrated in an ethanol series consisting of 30, 50, 75, 90 and 95% ethanol for 15 minutes each at 4° C. followed by three incubations in 100% ethanol for 20 minutes each at 4° C. The sample was then infiltrated in a 2:1 mixture of 100% ethanol:Unicryl™ resin (for 30 minutes followed by a 1:2 mixture of 100% ethanol:Unicryl™ resin (BBI Solutions) for 30 mins. Finally, the pellet was transferred to a BEEM capsule (Agar Scientific) and infiltrated in complete Unicryl™ resin overnight at 4° C. The resin was cured using light-polymerisation for 48 h by illumination from the underside of the BEEM capsules from a 12 V, 100 W type 6834 Philips projection lamp at a distance of 35 cm. Ultrathin sections (70 nm) were taken using a diamond knife on a Leica EM UC7 ultramicrotome fitted with a Leica M80 microscope (Leica Microsystems) and placed on 300 Mesh Hexagon Nickel 3.05 mm grids (Agar Scientific Ltd) before proceeding with immunogold labelling.
Immunogold Labelling TEM
[0172] A modified PBS (pH 8.2) containing 1% BSA, 500 μl/L Tween-20, 10 mM Na-EDTA and 0.2 g/L NaN.sub.3 (termed PBS+) was used throughout the following procedures for all dilutions of antibodies and gold probes. The ultrathin sections were initially blocked using normal goat serum (diluted 1:10 dilution in PBS+; Sigma-Aldrich) for 30 minutes at room temperature and then incubated with anti-Alexa Fluor® 488 primary antibody (1:50) (Thermo Scientific). The sections were washed three times in PBS+ for 2 minutes each followed by incubation with 10 nm gold particle-conjugated goat anti-rabbit IgG secondary probe at a 1:10 dilution in PBS+ for 1 h at room temperature. The sections were washed three times in PBS+ for 10 minutes each and four times in distilled water for 5 minutes each. Immunogold-labelled thin sections were subsequently post-stained in 2% (w/v) uranyl acetate for 1 h before imaging on the TEM.
Image Analysis
[0173] FIJI (https://fiji.sc) imaging processing package (J. Schindelin et al., Nat. Methods, vol. 9, no. 7, pp. 676-682, (2012)) was used for all image analysis. For quantification of fluorescence intensities, images were Z-projected to maximal intensity. Five to ten fields of view were taken from each condition and an average of 50 cells per condition were subjected to analysis. Firstly, a region of interest (the cell body) was drawn around an individual cell, excluding cells that had fused nuclei. Area-integrated intensity and mean grey value were measured as well as three selections from around the cell with no fluorescence (background). The corrected total cell fluorescence (CTCF) was then calculated using the equation:
CTCF=Integrated Density−(Area of selected cell×Mean fluorescence of background readings)
For quantification of internalised dGAE-488, a focal plane from the middle of the cell that contained maximal DAPI fluorescence was selected and subjected to analysis.
Data Analysis
[0174] Data and statistical analyses were performed using Microsoft Excel and GraphPad Prism 7. All data are expressed as the mean±standard error of the mean (SEM). When comparing two groups, Student's unpaired t-test with Welch's correction was used to determine statistical significance. When comparing more than two groups, one-way analysis of variance (ANOVA) with Dunnet's post-hoc test was used to determine differences between experimental groups and a control group. Differences were considered to be statistically significant if p<0.05.
Example 1: Examination of Formation of Fibrils by dGAE and dGAE-488
[0175] Soluble dGAE was fluorescently labelled using an Alexa-fluor 488 tag (dGAE-488) to enable internalisation of dGAE assemblies to be monitored and to distinguish exogenous dGAE from endogenously expressed tau. On average, there were 0.1 moles of 488 dye per mole of protein after every labelling reaction. The Alexafluor-tag labels amino groups at the N-terminus and on lysine residues, and hence we examined the tagged and untagged dGAE using TEM, SDS-PAGE and CD spectroscopy to determine whether the label affects aggregation and/or filament formation. Using the non-reducing conditions for aggregation established previously (Al-Hilaly et al., J. Mol. Biol., vol. 429, no. 23, pp 3650-3665 (2017)), dGAE and dGAE-488 produced morphologically similar short twisted fibrils (
Example 2: Study on Induction of Cell Death by dGAE
[0176] Aggregated dGAE (100 μM agitated for 72 h) and soluble dGAE (100 μM 0 h, no agitation) were applied at a concentration of 1 μM directly to dSH-SY5Y cells and incubated for 24 h. Cell viability was measured after 24 h using the ReadyProbes® assay to measure cell death (
Example 3: Internalisation of dGAE-488
[0177] The uptake of soluble and aggregated dGAE into dSH-SY5Y cells was investigated in order to examine whether aggregation state affects the efficiency of internalisation. The labelled dGAE-488 form was used to permit uptake to be visualised and measured having corrected for total cell fluorescence. Soluble or aggregated (72 h) dGAE-488 was incubated with dSH-SY5Y cells at 1 μM for 24 h. Confocal microscopy analysis showed that following exposure to soluble dGAE-488, fluorescence was observed within the cells as punctate staining (
Example 4: Phosphorylation Changes Following dGAE Internalisation
[0178] The results presented herein suggest that incubation with aggregated dGAE leads to significant cell death after incubation for only 24 h, whereas soluble dGAE is internalised but does not appear to be significantly toxic following 24 h incubation. Therefore, it was further investigated whether internalised soluble dGAE-488 may be capable of altering the aggregation or phosphorylation state of endogenous tau. Following exposure to soluble 1 μM dGAE-488, cells were fixed and immunolabelled using antibodies recognising tau epitopes outside of the dGAE sequence allowing specific detection of only endogenous tau (
Example 5: Effects of Exposure of Cells to Soluble dGAE-488
[0179] Western blots of the whole cell lysates were performed to examine the effect of exogenously applied dGAE on endogenous tau phospho-epitopes. Comparison of cells incubated with soluble dGAE with buffer-treated control cells revealed no change in levels of total tau and marginal increases in AT8 or AT180 immunoreactivity that did not reach statistical significance. While western blots measure total levels of phospho-tau in cells, immunofluorescent imaging is able to highlight subcellular localisation of any increases. In order to determine the effects of internalised soluble dGAE-488 on the solubility of endogenous tau in neurons, we undertook sequential extraction of tau protein from cell lysates following solubilisation with 1% Triton X-100 and centrifugation (16,000 g for 30 minutes). Following incubation with soluble dGAE for 24 h, there was a clear redistribution of tau protein immunolabelled with AT180 from Triton-X 100-soluble to Triton-X 100-insoluble fractions following incubation with soluble dGAE compared with buffer-treated controls. dGAE incubation produced a reduction in soluble tau immunoreactive with AT180 (83.0±5.5%, p=0.0201) and an increase in the insoluble form (139.4±14.5%, p=0.0352) (
Example 6: Localisation of Internalised dGAE-488
[0180] Intracellular fluorescent punctate particles were observed within the cytoplasm, close to the nucleus after 24 h of exposure of neurons to soluble dGAE-488. This staining pattern was consistent with its localisation in cytosolic vesicles. We therefore examined whether internalised dGAE-488 was localised to endosomal/lysosomal compartments. Soluble dGAE-488 was incubated with cells for 24 h and stained with LysoTracker®, a dye that labels acidic organelles (lysosomes and late endosomes), for 30 mins and then fixed and visualised by confocal microscopy. There was a significant increase in the intensity of the LysoTracker® fluorescence in cells exposed to soluble dGAE-488 compared to buffer-treated control cells (211.9±9.9%, p<0.0001;
Example 7: Ultrastructure of Accumulated Tau and dGAE-488 in Cells
[0181] TEM was used to examine the ultrastructure of accumulated tau and dGAE-488 in cells. dSH-SY5Y were exposed to 10 μM soluble dGAE-488 for 24 h and were processed for immunogold TEM using an antibody against Alexa Fluor® 488 to label dGAE-488 specifically. Vesicular structures and mitochondria were observed within the sectioned cells confirming that the TEM protocol preserved the cellular structure (
Example 8: Testing of Agents for Effects on Action of Aggregates of Tau Protein in Neuronal Cells
[0182] SHSY5Y cells are plated in 24 well plates (30,000 cells per well) and either differentiated using retinoic acid in depleted serum medium, as described previously, or left undifferentiated. Cells are treated in one of the following ways; either (1) pre-incubated in the presence of an agent of interest prior to adding tau aggregates or (2) co-incubated with an agent of interest and tau aggregates administered at the same time. Times for incubation are 24 hours at each stage meaning the total time for (1) is 48 hours and 24 hours for (2). dGAE-488 (1 μM) incubated with cells for 24 hours is sufficient for internalised protein to be seen. Following incubation, cells are photographed and analysis of images performed using ImageJ to quantify the fluorescence intensity within the cell body to provide a measure of dGAE-488 internalisation. The number of cells is counted to express the level of internalisation as mean fluorescence intensity/cell to take into account different numbers of cells in fields of view. Fragments of dGAE-488 are added at 1 μM. Agents for screening can be tested initially at a single concentration of 10 μM; those showing inhibitory activity are then tested over at a range of concentrations from 2 μM down to 20 nM and the activity of compounds reported as the concentration at which 50% inhibition of activity is observed. The activities to be measured include: (i) internalisation of tau measured by intracellular fluorescence; (ii) intracellular aggregation of tau measured by harvesting cells, lysing them and separating aggregated tau by ultracentrifugation and measuring aggregated tau by immunoblot with anti-tau antibody; and (iii) cytotoxicity measured using the ReadyProbes® assay to measure cell death (
Example 9: Tau Aggregation Inhibition Assays In Vitro
[0183] dGAE protein was diluted into 10 mM phosphate buffer, pH 7.4 (PB) with monoclonal antibody s1 D12 at a ratio of 4:1 protein:Ab (100 μM dGAE+25 μM s1D12 and 10 μM dGAE+2.5 μM s1D12) or 1:1 (25 μM dGAE+25 μM s1D12).
[0184] “s1D12” refers to a monoclonal antibody disclosed in UK application no. GB2010652.2 filed on 10 Jul. 2020, and an international (PCT) application filed on 9 Jul. 2021 in the name of WisTa Laboratories Ltd, both of which are hereby incorporated by reference in their entirety. The epitope bound by the CDRs of the specific binding molecule referred to as “S1D12” herein may be within an amino acid sequence comprising residues 337 to 349 of SEQ ID NO: 1 or preferably within an amino acid sequence comprising residues 341 to 353 of SEQ ID NO: 1. The epitope may comprise the amino acid sequence of SEQ ID NO: 8 (VEVKSEKLDFKDR). S1D12 comprises the CDRs VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3, wherein each of said CDRs comprises an amino acid sequence as follows: [0185] VHCDR1 comprises the sequence set forth in SEQ ID NO: 9 (NNAVG); [0186] VHCDR2 comprises the sequence set forth in SEQ ID NO: 10 (GCSSDGTCYYNSALKS); [0187] VHCDR3 comprises the sequence set forth in SEQ ID NO: 11 (GHYSIYGYDYLGTIDY); [0188] VLCDR1 comprises the sequence set forth in SEQ ID NO: 12 (SGSSSNVGGGNSVG); [0189] VLCDR2 comprises the sequence set forth in SEQ ID NO: 13 (DTNSRPS); and [0190] VLCDR3 comprises the sequence set forth in SEQ ID NO: 14 (VTGDSTTHDDL).
[0191] s1D12 may comprise:
TABLE-US-00007 (a) A VH domain comprising the sequence set forth in SEQ ID NO: 15 (QVQLQESGPSLVKPSQTLSLTCTVSGFSLNNNAV GWVRQAPGKVPESLVGCSSDGTCYYNSALKSRLDI TRDTSKNQISLSLSSVTTDDAAVYYCTRGHYSIYG YDYLGTIDYWGPGLLVTVSS); and/or (b) a VL domain comprising the sequence set forth in SEQ ID NO: 16 (QAVLTQPSSVSGSLGQRVSITCSGSSSNVGGGNS VGWYQHLPGSGLKTIIYDTNSRPSGVPDRFSGSRS GNTATLTINSLQAEDEGDYYCVTGDSTTHDDLVGS GTRLTVLG);
or a humanized variant of either thereof.
[0192] s1D12 may comprise:
TABLE-US-00008 (a) A heavy chain comprising the sequence set forth in SEQ ID NO: 17 (QVQLQESGPSLVKPSQTLSLTCTVSGFSLNNNAV GWVRQAPGKVPESLVGCSSDGTCYYNSALKSRLDI TRDTSKNQISLSLSSVTTDDAAVYYCTRGHYSIYG YDYLGTIDYWGPGLLVTVSSAKTTAPSVYPLAPVC GDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGV HTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVA HPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGG PSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP IQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGS VRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDI YVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRV EKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK); and/or (b) a light chain comprising the sequence set forth in SEQ ID NO: 18 (QAVLTQPSSVSGSLGQRVSITCSGSSSNVGGGNS VGWYQHLPGSGLKTIIYDTNSRPSGVPDRFSGSRS GNTATLTINSLQAEDEGDYYCVTGDSTTHDDLVGS GTRLTVLGGQPKSSPSVTLFPPSSEELETNKATLV CTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQS NNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVE KSLSRADCS);
or a humanized variant of either thereof.
[0193] As a positive control, dGAE was prepared at a final concentration of 10, 25 or 100 μM in 10 mM PB. Negative controls consisted of dGAE with a non-tau IgG antibody at a ratio of 4:1 (10 μM dGAE+2.5 μM anti-ovalbumin), or antibody alone (25 or 2.5 μM s1D12 and 2.5 μM anti-ovalbumin). Samples were agitated at 700 rpm at 37° C. for 3 days.
[0194] Transmission electron microscopy (TEM), circular dichroism (CD) and Thioflavin S (ThS) assays were performed as detailed in Al-Hilaly et. al. (2018) J. Mol. Biol. 430, 4119-4131. Briefly, TEM grids were prepared by adding 4 μL of sample to a carbon-coated grid followed by a wash with milli-Q filtered water then staining twice with 2% uranyl acetate. Grids were air dried and then imaged using a JEOL electron microscope operating at 80 kV.
[0195] For CD, 60 μL sample was placed in a 0.1 mm quartz cuvette and placed into a JASCO spectropolarimeter. 100 μL ThS in 20 mM MOPS buffer was added to 50 μL of each sample to a final concentration of 20 μM, mixed well, incubated at room temperature for 10 minutes then fluorescence intensity measured in a Cary Eclipse spectrophotometer using an excitation wavelength of 440 nm. Baseline readings from 10 mM PB were subtracted from CD and ThS measurements.
[0196] The findings from these three experiments indicate that s1D12 inhibits the assembly of dGAE into fibrils in vitro.
[0197] Firstly, dGAE fibrils were readily observed by TEM in samples using concentrations as low as 10 μM; at the same concentration in the presence of s1D12, no fibrils were present suggesting inhibition of assembly. Fibrils were present when using the same molar ratios of dGAE:Ab with a non-tau antibody, showing that the inhibitory effect is specific.
[0198] Circular dichroism reports on secondary structure characteristics of proteins in solution. Although 10 and 25 μM dGAE were too low in concentration to be observed using CD, and the signal is dominated by the characteristic 3-sheet signal expected from the antibody structure. At 100 μM dGAE, a random coil confirmation is revealed when the antibody CD signal is subtracted from the dGAE signal. This further indicates that for s1D12:dGAE at a ratio of 4:1, dGAE cannot assemble into 3-sheet rich fibrils.
[0199] Finally, Thioflavin S was used to report on the presence of fibrils. ThS is a dye that binds to amyloid, the underlying structure in dGAE fibrils, and fluoresces at a characteristic wavelength of around 483 nm when excited with light of a wavelength of 440 nm. A positive signal was clearly observed for 25 and 100 μM dGAE fibrils. However, when incubated with s1D12 at ratios of either 4:1 or 1:1, this signal was abolished, supporting the previous observations that no fibrils are present when dGAE is incubated with s1D12.
[0200] Collectively, these results provide evidence that (i) s1D12 specifically inhibits the assembly of dGAE into fibrils and (ii) these assays can be utilized to determine the inhibitory activity of other antibodies in disrupting formation of fibrils that strongly resemble the paired helical filaments that are present in the tau pathology characteristic of Alzheimer's disease.