Method for recovering tau seed in interstitial fluid from living body

Abstract

The present invention pertains to a method for recovering a tau seed in an interstitial fluid from a living body by a microdialysis method using a probe, the method comprising perfusing a perfusate through a probe and causing the interstitial fluid to flow into the probe. The cut-off value of the probe is 2 MDa or more, and the flow rate of the perfusate is 0.05-1.0 μL / min.

Description

Method for recovering tau seeds from brain interstitial fluid in living organisms

[0001] The present invention relates to a method for recovering tau seeds in brain interstitial fluid from a living body, and also to a method for detecting tau seeds in brain interstitial fluid recovered from a living body and a method for predicting the amount of tau protein accumulation in the brain.

[0002] Neurodegenerative diseases such as Alzheimer's disease have diverse pathogenic mechanisms, and research and development of therapeutic drugs based on these mechanisms is anticipated. Tau protein, a microtubule-associated protein present in nerve axons, is thought to be a major factor involved in the onset of neurodegenerative diseases. Indeed, tau protein aggregates and accumulates in the brains of patients with neurodegenerative diseases such as Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, and Pick's disease. Furthermore, it is known that the amount of tau protein accumulation correlates with cognitive decline, making tau protein a promising therapeutic target for neurodegenerative diseases.

[0003] In the tau protein aggregation reaction, it is known that tau protein aggregates generated during the progression of neurodegenerative diseases convert the normal tau protein structure into an abnormal form, promoting aggregation and thereby propagating tau protein aggregates (Non-Patent Document 1). Tau that converts the normal tau protein structure into an abnormal form and promotes aggregation is called tau seeds. Tau seeds propagate from neuron to neuron, so the amount of extracellular tau seeds serves as an indicator of tau seed propagation.

[0004] Non-Patent Document 2 describes the recovery of high molecular weight proteins from cerebral interstitial fluid by in vivo microdialysis.

[0005] SANDERS, David W., et al. Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron, 2014, 82.6: 1271-1288.YAMADA, Kaoru. In vivo microdialysis method to collect large extracellular proteins from brain interstitial fluid with high-molecular weight cut-off probes. JoVE (Journal of Visualized Experiments), 2018, 139: e57869.

[0006] The problem to be solved by the present invention is to provide a method for recovering tau seeds in brain interstitial fluid from a living body.

[0007] As a result of extensive research, the present inventors have discovered a method for recovering tau seeds in brain interstitial fluid from a living body, and have completed the present invention.

[0008] That is, the present invention includes the following embodiments. [1] A method for recovering tau seeds in brain interstitial fluid from a living organism by microdialysis using a probe, comprising: flowing brain interstitial fluid into a probe, wherein the cutoff value of the probe is 2 MDa or more, and the flow rate of the perfusion solution is 0.05 μL / min or more and 1.0 μL / min or less. [2] The method according to [1], wherein the perfusion solution contains BSA at a concentration of 0.1% to 1%. [3] The method according to [1], wherein the living organism is a mouse or a rat. [4] The method according to [1], wherein the living organism is an animal model of Alzheimer's disease. [5] The method according to [1], wherein the tau seeds are a complex containing 3 to 60 tau proteins. [6] A method for detecting tau seeds in brain interstitial fluid recovered from a living organism, comprising measuring tau seeds recovered by the method according to any one of [1] to [5] using a FRET method or a real-time QUIC method. [7] A method for predicting the amount of tau protein accumulation in the brain, comprising: detecting tau seeds in brain interstitial fluid collected from a living body by the method described in [6]; and predicting the amount of tau protein accumulation in the brain from the detected amount of tau seeds in the brain interstitial fluid.

[0009] According to the present invention, a method for recovering tau seeds in brain interstitial fluid from a living body can be provided. Also, according to the present invention, a method for detecting tau seeds in brain interstitial fluid recovered from a living body can be provided. Furthermore, according to the present invention, a method for predicting the amount of tau protein accumulation in the brain can be provided.

[0010] 1 is a diagram showing an example of a method for connecting tubes in a microdialysis method. 2 is a diagram showing the results of measuring FRET signals for cells of an example and a comparative example. 3 is a diagram showing the correspondence relationship between the amount of tau seeds in mouse ISF and the PHF1-positive area rate in the mouse brain.

[0011] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the present invention is not limited to the following examples.

[0012] The method according to this embodiment is a method for recovering tau seeds in brain interstitial fluid from a living body by microdialysis using a probe, the method including perfusing a perfusion fluid through the probe to cause the brain interstitial fluid to flow into the probe, wherein the cutoff value of the probe is 2 MDa or more and the flow rate of the perfusion fluid is 0.05 μL / min or more and 1.0 μL / min or less.

[0013] (Tau seeds) Tau seeds are aggregates of tau proteins. In vivo, tau seeds generally exist as aggregates of three or more tau proteins. Tau seeds are known to contain aggregates of 20 or more tau proteins (MIRBAHA, Hilda, et al. Tau trimers are the minimal propagation unit spontaneously internalized to seed intracellular aggregation. Journal of biological chemistry, 2015, 290.24: 14893-14903.), and some are known to contain aggregates of approximately 40 tau proteins (MAEDA, Sumihiro, et al. Granular tau oligomers as intermediates of tau filaments. Biochemistry, 2007, 46.12: 3856-3861.). The tau seeds recovered by the method of this embodiment are preferably aggregates of 3 to 60 tau proteins, more preferably aggregates of 20 to 60 tau proteins. The molecular weight of tau proteins is 55 to 62 kDa (HIROKAWA, Nobutaka; SHIOMURA, Yoko; OKABE, Shigeo. Tau proteins: the molecular structure and mode of binding on microtubules. The Journal of cell biology, 1988, 107.4: 1449-1459.). Therefore, the molecular weight of the tau seeds recovered by the method of this embodiment is preferably 3 MDa or less, more preferably 1 to 3 MDa.

[0014] Tau seeds have the ability to promote neurofibrillary tangle formation of tau protein (e.g., (1) YAMADA, Kaoru. Extracellular tau and its potential role in the propagation of tau pathology. Frontiers in neuroscience, 2017, 11: 314325. (2) FROST, Bess; JACKS, Rachel L.; DIAMOND, Marc I. Propagation of tau misfolding from the outside to the inside of a cell. Journal of biological chemistry, 2009, 284.19: 12845-12852. (3) GOEDERT, Michel; EISENBERG, David S.; CROWTHER, R. Anthony. Propagation of tau aggregates and neurodegeneration. Annual review of neuroscience, 2017, 40: 189-210.). Tau seeds are substances containing tau protein, including oligomers, monomers, complexes thereof, and other forms of tau protein, provided that they have the ability to cause fibrillar tau protein to appear in cultured cells or animal brains.

[0015] (Microdialysis Method) Microdialysis is a method in which a probe having a semipermeable membrane is inserted into a living body, perfusion is performed at a constant rate within the probe, and substances present between cells are collected into the perfusion fluid through the semipermeable membrane.

[0016] The living body is a non-human animal, preferably a mammal, more preferably a mouse or a rat. The in vivo state may be within the brain of such an animal, preferably within the brain parenchyma of such an animal.

[0017] The probe used in the microdialysis method has (1) an inlet tube that introduces a perfusion fluid from the outside to the living body, (2) an outlet tube that discharges the dialyzed fluid from the living body to the outside, and (3) a semipermeable membrane that is inserted into the living body and dialyzes substances in the living body.

[0018] A commercially available probe may be used. The cutoff value of the probe is 2 MDa or more, preferably 2.5 MDa or more, and more preferably 3 MDa or more. The cutoff value of the probe may be selected to be about 3 MDa.

[0019] The perfusion fluid is not particularly limited, and may be artificial cerebrospinal fluid (aCSF). From the viewpoint of recovery efficiency of the target substance, BSA may be added to the aCSF. The concentration of BSA is preferably 4% or less, more preferably 0.05% to 2% or less, even more preferably 0.1% to 1% or less, even more preferably 0.2% to 1% or less, and may be 0.4% to 0.6% or less.

[0020] During perfusion, the flow rate of the perfusion solution is preferably maintained constant. The flow rate may be controlled by a known method, for example, by using a syringe pump to allow the perfusion solution to flow from a syringe at a constant flow rate. The perfusion flow rate is 0.05 μL / min to 1.0 μL / min, preferably 0.06 μL / min to 0.3 μL / min, more preferably 0.07 μL / min to 0.15 μL / min, and even more preferably 0.08 μL / min to 0.12 μL / min.

[0021] The microdialysis method includes a step of inserting a guide cannula into the brain interstitium. The insertion site and insertion angle may be determined by a known method, for example, based on a brain atlas. The material of the guide cannula is not particularly limited and may be, for example, metal or glass. A commercially available guide cannula may be used, for example, an MAB5 guide cannula (manufactured by Microbiotech) or an AG-X guide cannula (manufactured by Eicom). The method of inserting the guide cannula into the brain interstitium may be performed by a known method.

[0022] After the insertion of the guide cannula, a period may be provided for the tissue to recover from the damage caused by the insertion. The period is not particularly limited, but is, for example, 12 hours or more, preferably 1 day or more, and more preferably 3 days or more.

[0023] The microdialysis method further includes a step of connecting the probe, the perfusion fluid supply device, and the collection device to form a flow path. The connection is not particularly limited as long as it is a device capable of forming a flow path, and for example, FEP tubing may be used. The length of the flow path may be adjusted as appropriate. The perfusion fluid supply device is not particularly limited as long as it is a device that can maintain a constant flow rate of the perfusion fluid, and may be, for example, a device in which a syringe filled with perfusion fluid is set in a syringe pump. The collection device is not particularly limited as long as it is a device that can collect the dialysis fluid after perfusion, and may include, for example, a fraction collector.

[0024] To prevent loss of perfusion fluid due to internal pressure built up in the probe, it is preferable to use both a pump that introduces perfusion fluid into the probe and a pump that collects dialysate from the probe. If the probe is provided with a pressure-canceling vent, only the pump that collects dialysate from the probe may be used. Each pump may be a known or commercially available pump; for example, a syringe pump may be used as the pump that introduces perfusion fluid into the probe, and a roller pump may be used as the pump that collects dialysate from the probe. When a roller pump is used, it is preferable to use a roller tube as the tube installed in the pump.

[0025] From the viewpoint of preventing nonspecific binding of proteins to the tubing, the microdialysis method preferably includes a step of blocking the tubing before the step of connecting the probe, the perfusion fluid supply device, and the collection device to form a flow path. Blocking may be performed by a known method, for example, using BSA. From the same viewpoint, it is preferable to use an instrument with low protein adsorption in the step of collecting dialysate from the probe.

[0026] The probe is preferably perfused with a perfusion solution before being inserted into the cerebral interstitium. The perfusion time is not particularly limited and may be, for example, from 10 minutes to 2 hours, from 15 minutes to 1 hour, or from 20 minutes to 40 minutes. The flow rate of the perfusion solution is not particularly limited and may be, for example, from 0.05 μL / min to 10 μL / min, from 0.5 μL / min to 6.0 μL / min, or from 1.0 μL / min to 3.0 μL / min.

[0027] The microdialysis method includes a step of recovering the perfused solution. The recovery time may be adjusted as appropriate and may be continued until the required amount of sample is recovered. From the viewpoint of the stability of the cerebral interstitial fluid (ISF), it is preferable to start the recovery after a certain time has elapsed after the flow rate of the perfusate is set to a desired value. The time to start the recovery may be, for example, 8 hours, preferably 12 hours, more preferably 16 hours, and even more preferably 24 hours after changing the flow rate.

[0028] The solution collected by microdialysis may be purified or concentrated. The purification or concentration step may be performed by a known method, such as centrifugation, sucrose density gradient centrifugation, or chromatography.

[0029] The solution collected by microdialysis may be frozen and stored. The freezing step may be carried out by a known method, for example, by gently freezing in a freezer or by rapidly freezing using liquid nitrogen.

[0030] When tau seeds are recovered by homogenizing a brain sample after the death of a non-human animal, it is impossible to determine whether the tau seeds originate from inside or outside the cells. However, the method of this embodiment makes it possible to recover tau seeds from brain interstitial fluid in a living body.

[0031] (Method for detecting tau seeds) The method according to this embodiment further includes a method for detecting tau seeds in brain interstitial fluid collected from a living body, which includes measuring tau seeds by the FRET method or the real-time QUIC method.

[0032] The method for detecting tau seeds is not particularly limited, and examples thereof include the FRET (Fluorescence Resonance Energy Transfer) method and the real-time QUIC (real-time queaking induced conversion) method.

[0033] The solution recovered by the above-mentioned process may be subjected to a detection method without further treatment. Alternatively, the recovered solution may be subjected to a detection method after purification, concentration, or dilution. The purification, concentration, or dilution process may be performed by a known method.

[0034] FRET Method FRET is a phenomenon in which excitation energy is transferred non-radiatively by resonance from an excited donor fluorescent protein (energy donor) to an acceptor fluorescent protein (energy acceptor).

[0035] The donor fluorescent protein constituting the donor fluorescent protein region may be a fluorescent protein commonly used to induce FRET. Examples of donor fluorescent proteins include CFP, mTFP1, CyPet, ECFP, SECFP, mTurquoise, and mTurquoise 2. The donor fluorescent protein may be a full-length sequence or a partial sequence, as long as it can induce FRET.

[0036] The acceptor fluorescent protein constituting the acceptor fluorescent protein region may be a fluorescent protein commonly used to induce FRET. Examples of acceptor fluorescent proteins include YFP, Ypet, EYFP, Venus, and mNeonGreen. The acceptor fluorescent protein may be a full-length sequence or a partial sequence, as long as it can induce FRET.

[0037] Preferred combinations of donor fluorescent proteins and acceptor fluorescent proteins include CFP and YFP, mTurquoise2 and mNeonGreen, SECFP and YPet, ECFP and Venus, ECFP and YPet, Turquoise and Venus, and Turquoise and YPet.

[0038] Detection of tau seeds by the FRET method may be performed by a known method using known or commercially available tau biosensor cells. In the step of detecting tau seeds by the FRET method, tau seeds may be added to tau biosensor cells expressing tau proteins bound to donor / acceptor fluorescent proteins. Tau seeds may be detected by detecting a FRET signal generated when tau proteins approach each other after tau protein aggregation is promoted.

[0039] Real-time QUIC method In the real-time QUIC method, a mixture of collected tau seeds and a substrate, recombinant tau protein, is sonicated or shaken to promote the aggregation of tau protein, thereby detecting the aggregation of tau protein and detecting the presence of tau seeds (METRICK, Michael A., et al. A single ultrasensitive assay for detection and discrimination of tau aggregates of Alzheimer's and Pick diseases. Acta neuropathologica communications, 2020, 8: 1-13.). Because this method amplifies tau protein aggregates, it may also be used to detect trace amounts of tau seeds.

[0040] Detection of tau seeds by the real-time QUIC method may be carried out by a known method.

[0041] The amount of tau seeds in ISF may be analyzed by a known method, for example, based on the intensity of the fluorescent signal detected by the above-mentioned method. The fluorescent signal intensity may be measured using a known method and a commercially available device. In the case of the FRET method, the fluorescent signal to be measured is the fluorescent signal of an acceptor fluorescent protein.

[0042] (Method for predicting the amount of tau protein accumulation in the brain) The method according to this embodiment further includes a method for predicting the amount of tau protein accumulation in the brain, which comprises detecting tau seeds in brain interstitial fluid collected from a living body, and predicting the amount of tau protein accumulation in the brain from the detected amount of tau seeds in the brain interstitial fluid.

[0043] In this embodiment, predicting the amount of tau seeds accumulated in the brain from the detected amount of tau seeds in the brain interstitial fluid may involve treating the amount of tau seeds in the brain interstitial fluid as a relative value and predicting a relative change in the amount of tau protein accumulated in the brain. Also, predicting the amount of tau protein accumulated in the brain from the detected amount of tau seeds in the brain interstitial fluid may involve predicting the amount of tau protein accumulated in the brain by using calibration data.

[0044] As calibration data for intracerebral tau protein, data obtained by a known method may be used, for example, the PHF1-positive area ratio of a brain section calculated by immunostaining using a PHF1 antibody, which is a phosphorylated antibody against tau protein. The PHF1 antibody is a phosphorylated antibody against tau protein and is a monoclonal antibody that recognizes serine 396 and serine 404. The PHF1-positive area ratio may be determined, for example, by calculating the PHF1-positive area in the hippocampus and dividing it by the area of ​​the hippocampus. In the method for predicting intracerebral tau protein accumulation according to this embodiment, a relative change in intracerebral tau protein accumulation may be predicted from a change in the amount of tau seeds in brain interstitial fluid. The relative change in intracerebral tau protein accumulation may be understood as a change in a calibration curve created from the amount of tau seeds in brain interstitial fluid and the PHF1-positive area ratio.

[0045] In the method according to the present embodiment, for example, a calibration curve may be prepared from the amount of tau seeds in brain interstitial fluid and the PHF1-positive area ratio measured in advance, and the detected amount of tau seeds in brain interstitial fluid may be converted to a PHF1-positive area ratio using the calibration curve. Furthermore, a calibration curve may be prepared from the PHF1-positive area ratio measured in advance and the amount of tau protein accumulation in the brain measured in advance by a known method, and the PHF1-positive area ratio obtained by the above conversion may be converted to the amount of tau protein accumulation in the brain using the calibration curve.

[0046] In the method according to this embodiment, for example, a calibration curve may be created using the amount of tau seeds in brain interstitial fluid measured in advance and the amount of tau protein accumulation in the brain measured in advance by a known method, and the detected amount of tau seeds in the brain interstitial fluid may be converted to the amount of tau protein accumulation in the brain using the calibration curve.

[0047] The method according to this embodiment may include, for example, detecting the amount of tau seeds in brain interstitial fluid over time and analyzing the changes therein to predict the changes in the amount of tau protein accumulated in the brain over time.

[0048] Some examples of the above-described embodiments of the present invention will be described below. The present invention will be specifically explained below using examples, but the present invention is not limited to these examples.

[0049] (Example 1: Insertion of a guide cannula into brain coordinates (the hippocampus was selected as the target region. With Bregma as the origin, A / P: −2.8 mm, M / L: 0.5 mm, D / V: 1.3 mm, 38 degrees) predetermined using a brain atlas) P301S tau transgenic mice (PS19) aged 4-5 months, 8 months, and 12 months were used.

[0050] Mice were anesthetized by intraperitoneal administration of 400 mg / kg of chloral hydrate (Tokyo Chemical Industry Co., Ltd.). The hair on the mouse's head was shaved with a surgical shaver, and the mouse's head was fixed at three points to a mouse and neonatal rat adapter (Stoelting Co.) using ear bars (Stoelting Co.) and a nose clamp (Stoelting Co.).

[0051] The mouse was fixed in a stereotaxic mouse and neonatal rat adapter (Kopf), and the scalp was incised from the glabella to the midline using a scalpel. Blood and connective tissue on the skull were removed with a moist cotton swab.

[0052] A hole was drilled in the skull at approximately A / P: +1.5 mm, M / L: +1.0 mm using a drill (manufactured by Foredom Electric Company), and a bone screw (manufactured by BASi) was embedded.

[0053] A drill was attached to the stereotaxic manipulator arm, and it was confirmed that the sagittal suture line and the stereotaxic AP direction were moved parallel to each other.

[0054] The drill was lowered until it lightly touched the Lambda and its ventral coordinate was recorded. This procedure was repeated for the Bregma to ensure that both vertical measurements were equal. If not, the height of the nose clamp was adjusted and the measurement was repeated.

[0055] The vertical coordinates of the drill were also recorded to confirm that the heights of both were equal. If they were not equal, the heights of the left and right ear bars were adjusted and the measurements were taken again.

[0056] The drill was moved to A / P: -2.8 mm, M / L: -0.5 mm, and then the drill was rotated to drill a hole slightly less than 1 mm in diameter in the skull until the brain surface became visible.

[0057] An MAB5 guide cannula (Microbiotech) was fixed to the stereotaxic manipulator arm using a clamp. Just before this, it was confirmed that the dummy probe could be removed from the guide cannula.

[0058] The stereotaxic manipulator arm was tilted 38 degrees from the vertical, away from the brain, and the guide cannula was then moved to a point A / P: −2.8 mm, M / L: ±2.0 mm, and then lowered 1.3 mm from the brain surface and slowly inserted into the brain.

[0059] A circular lock piece was cut out from the underside of the lid of a 1.5 mL centrifuge tube with a utility knife to create a "crown," which was then set to contain the guide cannula and bone screw inside.

[0060] After confirming that no bleeding had occurred, dental cement (manufactured by 3M) was poured into the crown to completely cover both the metal part of the guide cannula and the bone screw, and the crown was left to stand for approximately 20 minutes until the dental cement had completely hardened.

[0061] After removing the guide cannula from the clamp, the mice were removed from the stereotaxic system and housed singly in cages. They were allowed to recover for one to several days before microdialysis.

[0062] Example 2: Collection of brain interstitial fluid (ISF) samples by in vivo microdialysis. 4% BSA / aCSF and 0.5% BSA / aCSF were prepared by diluting a 30% BSA solution with artificial cerebrospinal fluid (aCSF; 1.3 mM CaCl, 1.2 mM MgSO, 3 mM KCl, 0.4 mM KHPO, 25 mM NaHCO, 122 mM NaCl, pH 7.35).

[0063] The above two solutions were passed through a syringe filter unit with a pore size of 0.1 μm to prepare 4% BSA / aCSF (blocking solution) and 0.5% BSA / aCSF (perfusion solution).

[0064] The perfusion rate of microdialysis was measured. First, a 70 cm FEP tube was connected to one end of a roller tube (RT-5S, manufactured by Eicom Co., Ltd.) and a 70 cm FEP tube cut in half was connected to the other end using a 24 G roller tube joint (RTJ, manufactured by Eicom Co., Ltd.), forming an outlet line.

[0065] The roller tube portion of the outlet line was attached to a roller pump (ERP-10, manufactured by Eicom Co., Ltd.). The FEP tube portion cut in half was placed in distilled water and perfused for 100 minutes at a pump rate of 100. The pump rate corresponding to the flow rate used in the experiment (in this case, 10.0 μL / min, 1 μL / min, and 0.1 μL / min) was calculated from the recovered volume.

[0066] A 2.5 mL syringe equipped with a 23G blunt needle was filled with blocking solution and set in a syringe pump. A new FEP tube (inlet line) was connected to this, and the inlet line was filled with blocking solution by pumping the solution with the syringe pump. This inlet line was then connected to the outlet line using a roller tube joint. Perfusion was performed at 1.0 μL / min for approximately 3 hours, and the inside of the line was blocked with BSA.

[0067] A 0.5 mL gas-tight Hamilton syringe filled with perfusion solution was set on a syringe pump. The inlet line and outlet line, connected with a roller tube joint, were connected to a 0.5 mL gas-tight Hamilton syringe, and the tube was completely replaced with perfusion solution by pumping at 1.0 μL / min. Then, the joint connecting the inlet line and outlet line was removed, and the free end of the inlet was connected to the inlet port of a Microbiotech MAB5 probe (MWCO: 3 MDa, recommended membrane length 3 mm), and the free end of the outlet line FEP tubing was connected to the outlet port of the MAB5 probe (MWCO: 3 MDa, recommended membrane length 3 mm).

[0068] A 15 ml tube was filled with perfusion solution, the MAB5 probe was immersed in the solution, and perfusion was carried out in this state at 1.0 μL / min for about 30 minutes.

[0069] Mice that underwent guide cannulation surgery were anesthetized by intraperitoneal administration of 400 mg / kg chloral hydrate. After confirming that the anesthesia was sufficient, a mouse collar was attached and the dummy probe was carefully removed.

[0070] The sleeve protecting the dialysis probe was removed with great care so as not to touch the dialysis membrane, and the probe was slowly inserted through the guide cannula in the mouse's head, taking care not to touch the inner wall. The syringe pump and roller pump flow rates were set to 10.0 μL / min, and perfusion was performed for 10 minutes.

[0071] The junction of the guide cannula and the dialysis probe was fixed with dental cement.

[0072] The flow rates of the syringe pump and roller pump were set to 0.1 μL / min, and the free end of the outlet line was connected to a cooled fraction collector in which a sampling tube was set in order to collect an ISF sample.

[0073] The mouse was transferred to a cage in the return free-moving system and connected using a mouse collar. The return free-moving system was operated, allowing the mouse to move freely.

[0074] Once the required amount of ISF had been collected, the probe was removed, and the sample was transferred to a low-protein-binding tube using a low-protein-binding tip and stored at -80°C. All ISF samples in the following procedures were handled using low-protein-binding tips and tubes. After changing the flow rate to 0.1 μL / min to stabilize the ISF state, samples taken after the first 16 hours were used.

[0075] As a comparative example, a sample was prepared by carrying out the same steps as described above on a 12-month-old mouse, except that a probe with a MWCO of 1 MDa was used, and then collecting the sample.

[0076] (Example 3: Detection of tau seeds using tau biosensor cells) Cell seeding A slide chamber or the like for seeding cells was prepared, and a poly-D-lysine (PDL) solution was added to each well to coat them at 37°C for 2 hours.

[0077] After removing the PDL solution, the plate was washed three times with DPBS. Biosensor cells (TauRD P301S FRET Biosensor: ATCC #CRL-3275) were added at 1.0 × 105 The cells were suspended in DMEM / 10% FBS / 1% PS to a concentration of 100 cells / mL, and placed in a well with a bottom area of ​​1 cm. 2 The mixture was left to stand in a CO2 incubator at 37°C for 24 hours.

[0078] Addition of ISF sample The ISF sample frozen at -80°C was thawed on ice. All subsequent ISF samples were handled on ice.

[0079] In a clean bench, 5% of the ISF sample medium in each well was mixed with 0.1% of the P3000 reagent from the Lipofectamine 3000 Transfection Reagent kit (ThermoFisher Scientific), followed by 0.1% of the Lipofectamine 3000 reagent. The mixture was stirred for 10 seconds and then allowed to stand for 5 minutes.

[0080] The ISF sample was dropped into each well, and the plate was again left standing in the CO 2 incubator for 24 hours.

[0081] To avoid cytotoxicity of Lipofectamine 3000 and the ISF sample, the medium was removed 24 hours after the addition of the ISF sample, and an appropriate amount of DMEM / 10% FBS / 1% PS was promptly added. After the medium change, the cells were left to stand in a CO incubator for an additional 48 hours.

[0082] Fixation and nuclear staining of biosensor cells The medium was removed from each well, and 4% paraformaldehyde (manufactured by TAAB, paraformaldehyde, PFA solution) was added. The cells were then left to stand for 30 minutes at room temperature in the dark to fix the cells.

[0083] The 4% PFA solution was removed, and the cells were washed three times with PBS.

[0084] A 4% BSA / PBS solution was mixed with 0.1% DRAQ5 reagent, and an appropriate amount was added to each well, followed by leaving the plate to stand at room temperature under light-shielded conditions for 45 minutes.

[0085] The DRAQ5 solution was removed and the cells were washed three times with PBS.

[0086] The sections were mounted with an aqueous mounting medium, taking care not to mix in air bubbles, and left overnight at 4°C in the dark, after which the FRET signal was observed.

[0087] The results are shown in Figure 2. In 12-month-old mice in which tau seed formation is advanced, tau seeds were not detected by the conventional method, whereas tau seeds were detected by the method of this embodiment.

[0088] Example 4: Detection of tau protein accumulation in mouse brain In the same manner as in Examples 1 to 3, ISF was collected from the hippocampus of P301S tau transgenic mice (PS19), and the amount of tau seeds in the collected ISF was measured.

[0089] Visualization of Tau Protein Accumulation in Mouse Brains Paraffin sections were prepared from the brains of mice from which ISF had been collected, and immunostained using the following method.

[0090] The brain was placed in a PBS solution (pH 7.4) containing PFA at a final concentration of 4%, and shaken at room temperature for 24 hours for immersion fixation.

[0091] The fixed brains were divided into five coronal sections using a Brain Matrix (ASI). The divided brain samples from each individual were placed in a sample pack (Eiken Chemical Co., Ltd.) and then placed in 70% ethanol, 90% ethanol, and 100% ethanol (Fujifilm Wako Pure Chemical Corporation) and shaken at room temperature for 1 hour each. After shaking, the brain samples were transferred to 100% ethanol and dehydrated overnight at room temperature.

[0092] After dehydration by shaking, the brain samples were infiltrated with xylene (Fujifilm Wako Pure Chemical Industries, Ltd.) and shaken twice at room temperature for 2 hours to allow the xylene to penetrate the brain samples. The brain samples were then infiltrated with liquid paraffin (Fujifilm Wako Pure Chemical Industries, Ltd.) three times in a 67°C incubator. The final time, the brain samples were infiltrated overnight and embedded in paraffin to prepare paraffin blocks.

[0093] The obtained block was sliced ​​into 4 μm-thick slices using a microtome (manufactured by Zeiss) and attached to MAS-coated slide glasses (manufactured by Matsunami Glass Industry Co., Ltd.) The obtained brain slices were dried in a thermostatic bath at 37° C. for 96 hours.

[0094] After drying, the slides with the brain sections were deparaffinized by immersing them in xylene for 5 minutes three times, then in 100% ethanol, 100% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol, one minute each, and then washed with running tap water for 10 minutes.

[0095] The slide glass carrying the brain slice was immersed in boiling 10 mM citrate buffer (1.8 mM anhydrous citric acid (Fujifilm Wako Pure Chemical Industries, Ltd.), 8.2 mM anhydrous trisodium citrate (Fujifilm Wako Pure Chemical Industries, Ltd.) in DW, pH 6.0) for 10 minutes and then microwave-treated.

[0096] The buffer was cooled to room temperature at 4°C and washed with running tap water for 10 minutes.

[0097] The slide glass on which the brain slices were placed was incubated at 37°C for 6 minutes in PK solution (manufactured by Takara Bio Inc.) containing Tris buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.6, Tris buffered saline: TBS) at a final concentration of 100 μg / mL, and then washed with running water for 10 minutes.

[0098] As a blocking solution, 10% Calf cerium (manufactured by Life Technologies) diluted with PBS was placed on the brain slices and allowed to stand at room temperature for 30 minutes.

[0099] A PHF-1 antibody solution was prepared by diluting it 1000-fold with the blocking solution. The blocking solution on the brain slices was removed, and the PHF-1 antibody solution was placed on the brain slices and allowed to stand overnight at room temperature. After standing, the PHF-1 antibody solution on the brain slices was removed, and the brain slices were washed three times with TBS.

[0100] Biotinylated anti-mouse IgG antibody (Vector Laboratories) was dissolved in the blocking solution at a final concentration of 3 μg / mL to prepare a secondary antibody solution. The secondary antibody solution was placed on the brain slices and allowed to stand at room temperature for 2 hours. After standing, the secondary antibody solution on the brain slices was removed, and the slices were washed three times with TBS.

[0101] Using an elite ABC kit (Funakoshi Co., Ltd.) containing streptavidin-HRP, the reaction solution was placed on the brain slice to induce an avidin-biotin binding reaction. After standing at room temperature for 1 hour, the slice was washed three times with TBS.

[0102] The brain sections were immersed for 6 minutes in a solution prepared by adding 3,3'-Diaminobenzaminidine tetrahydrochloride (DAB, manufactured by DOJINDO LABORATORIES, final concentration 220 μg / mL) and hydrogen peroxide solution (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd., final concentration 0.012%) to TBS to develop color.

[0103] The brain slices were dehydrated and cleared by immersing them in 70% ethanol, 90% ethanol, 100% ethanol, and 100% ethanol in that order for 1 minute each, and then immersed in xylene for 1 minute three times in total, and then mounted in HSR solution (manufactured by Sysmex Corporation) to obtain mouse brain slice samples.

[0104] Quantitative Evaluation of Tau Protein Accumulation in Mouse Brain Mouse brain slice samples were observed under bright field conditions using an inverted optical microscope (BZ-X710, Keyence Corporation) and photographed with a CCD camera. To capture the entire coronal brain section, multiple sections of each slice were photographed in a Z-stack (960 × 720 pixels per image, tif file) and then tiled.

[0105] Using ImageJ, the hippocampal region of the tiled image was selected, the relevant portion was cropped, and the area of ​​the relevant region was calculated.

[0106] To measure the proportion of stained areas in the cropped region, the image was binarized. During binarization, a threshold was set using brain sections from wild-type mice with no tau accumulation as a negative control, and the area of ​​the region showing staining above the threshold was measured and defined as the stained area.

[0107] For each mouse, the PHF1-positive area was divided by the area of ​​that region to calculate the PHF1-positive area ratio. Figure 3 shows the correlation between the PHF1-positive area ratio, a phosphorylated antibody against tau protein, and the amount of tau seeds detected in ISF in the same mouse. It was suggested that there is a proportional relationship between the amount of tau protein accumulated in the mouse brain and the amount of tau seeds detected in the mouse ISF. It was found that it is possible to predict the amount of tau protein accumulated in the brain from the amount of tau seeds in the ISF.

Claims

1. A method for recovering tau seeds in brain interstitial fluid from a living body by microdialysis using a probe, the method comprising perfusing a perfusion fluid through the probe to allow brain interstitial fluid to flow into the probe, the cutoff value of the probe being 2 MDa or more, and the flow rate of the perfusion fluid being 0.05 μL / min or more and 1.0 μL / min or less.

2. The method of claim 1, wherein the perfusion solution contains BSA at a concentration of 0.1% to 1%.

3. The method of claim 1, wherein the living organism is a mouse or a rat.

4. The method according to claim 1, wherein the living body is an animal model of Alzheimer's disease.

5. The method of claim 1, wherein the tau seed is a complex containing 3 to 60 tau proteins.

6. A method for detecting tau seeds in brain interstitial fluid recovered from a living body, comprising measuring tau seeds recovered by the method according to any one of claims 1 to 5 using the FRET method or the real-time QUIC method.

7. A method for predicting the amount of tau protein accumulation in the brain, comprising: detecting tau seeds in brain interstitial fluid collected from a living body using the method of claim 6; and predicting the amount of tau protein accumulation in the brain from the detected amount of tau seeds in the brain interstitial fluid.

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