Method for screening for analgesic that inhibits tentonin 1 / tmem150a activity

Abstract

The present invention has identified a novel function of tentonin 1 and provides tentonin 1 as a target for treating acute and chronic pain. The present invention has confirmed that tentonin 1 is expressed in sensory Schwann cells as a mechanochannel and mediates slowly adapting currents in response to mechanical stimuli, and has confirmed analgesic effects against various pain-inducing stimuli in a tentonin 1 knockout animal model. Therefore, the present invention is expected to be used as a platform for developing analgesics which can be used for the treatment of inflammatory and neuropathic pain, and the like.

Description

Screening method for analgesic drugs that inhibit temtonin1 / TMEM150A activity

[0001] The present invention confirms that Tentonin 1 (Tentonin 1 / TMEM150a) is a mechanochannel that mediates slow-adapting currents and plays an important role in the pain transmission mechanism, and provides a cell or animal model overexpressing Tentonin 1 and a method for screening analgesics using the same.

[0002] Pain is a warning signal our bodies use to protect us from harmful stimuli or tissue damage. However, when pain is severe or prolonged, it can interfere with normal life. Acute pain, such as that caused by surgery, fractures, or bruises, is triggered by strong mechanical stimuli. While numerous genes targeting chronic pain have been discovered, those targeting acute mechanical pain remain unknown.

[0003] Mechanical pain is primarily mediated by mechanoreceptors, whose terminals are thought to penetrate the epidermis and form free nerve endings without specific receptor cells. However, this concept has recently been revised, and it has been reported that free nerve endings in the skin are surrounded by sensory Schwann cells (SSCs). These SSCs are a type of Schwann cell that surround nociceptive nerve endings in the epidermis and express Sox10. Sensory Schwann cells are known to mediate mechanical pain. For sensory Schwann cells to sense mechanical stimuli, there must be a molecular sensor that converts the mechanical stimulus into an electrical signal. In other words, a mechanochannel is required. However, it is not yet known which channel mediates mechanical pain in sensory Schwann cells.

[0004] Several types of ion channels, or mechanochannels, are known to open in response to mechanical stimulation. Among these, tentonin-3 is one such mechanochannel. When mechanical stimulation is applied to cells expressing tentonin-3, the channel rapidly opens and then closes slowly, distinguishing it from piezoelectric channels. Tentonin-3 has been identified as important in proprioception, blood pressure regulation (baroreceptor reflex), and insulin secretion. Tentonin-1 is known to be a homolog of tentonin-3. However, the function of tentonin-1 remains unknown.

[0005] The present invention was completed by conducting a thorough study on the function of tentonin 1, and confirming that tentonin 1 is intensively expressed in sensory Schwann cells, and that tentonin 1, like tentonin 3, is a mechanical channel and mediates mechanical pain in sensory Schwann cells.

[0006] The technical problem to be achieved by the present invention is to provide a cell and animal model expressing tenonin 1, and to provide a method for screening a substance inhibiting tenonin 1 activity or an analgesic using the cell or animal model.

[0007] In addition, the present invention aims to provide a mechanical stimulus-refractory animal model in which tentorin 1 is knocked out.

[0008] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0009] To solve the above problem, the present invention provides a method for screening for a substance that inhibits the activity of tensinogen 1, comprising the following steps:

[0010] (1) A step of preparing a cell or cell line expressing Tentonin 1;

[0011] (2) A step of treating the candidate substance to the above cells;

[0012] (3) A step of applying mechanical stimulation to the above cells;

[0013] (4) a step of measuring slow adaptation current in the above cell; and

[0014] (5) A step of selecting the candidate substance as a substance that inhibits the activity of tensinogen 1 when the slow adaptation current measured in the above cell is lower than the slow adaptation current of a cell that has not been treated with anything.

[0015] In addition, the present invention provides a method for screening an analgesic comprising the following steps:

[0016] (1) Step of preparing cells or cell lines expressing Tentonin 1;

[0017] (2) A step of treating the candidate substance to the above cells;

[0018] (3) A step of applying mechanical stimulation to the above cells;

[0019] (4) a step of measuring the activity level of tensinogen 1 in the above cells; and

[0020] (5) A method for screening an analgesic, comprising: a step of selecting the candidate substance as a pain relief substance when the activity of tentonin 1 in the above cells is lower than the activity of tentonin 1 in cells that have not been treated with anything;

[0021] As one embodiment of the present invention, the tentonin 1-expressing cells of each of the above methods include cells that overexpress or express tentonin 1, such as a cell line expressing tentonin 1 or a stable cell line.

[0022] As another embodiment of the present invention, the step (4) may be performed by using a calcium imaging technique or by measuring slow adaptation currents in the cells, and in a specific experiment of the present invention, the step was performed by measuring slow adaptation currents in the cells.

[0023] As another embodiment of the present invention, the step (5) may be to select the candidate substance as a pain relief substance when the slow adaptation current measured in the cell is lower than the slow adaptation current of a cell that has not been treated with anything.

[0024] As another embodiment of the present invention, in step (1) of each of the above methods, the cell may be a cell transformed with a vector containing a gene encoding tentonin 1, and the step may be performed by transforming the cell with a vector for expressing tentonin 1 and selecting the transformant.

[0025] As another embodiment of the present invention, the analgesic may be used to treat neuropathic pain.

[0026] In addition, the present invention provides a mechanical stimulus-refractory animal model in which tentorin 1 is knocked out or knocked down.

[0027] In the present invention, the mechanical stimulus refractory animal model refers to an animal with a reduced response to mechanical stimulation and has the characteristic of being insensitive to pain caused by mechanical stimulation.

[0028] In addition, the present invention provides an animal model that overexpresses tentinin 1.

[0029] In the present invention, the animal model overexpressing the above-mentioned tenonin 1 is hyperresponsive to mechanical stimulation and has the characteristic of being sensitive to pain caused by mechanical stimulation.

[0030] The animal model overexpressing the tentonin 1 of the present invention is sensitive to pain and thus exhibits a more dramatic effect on analgesics. Therefore, the animal model overexpressing tentonin 1 can be used for analgesic screening.

[0031] Accordingly, the present invention provides a method for screening an analgesic comprising the following steps:

[0032] (a) a step of preparing the above-described tentorin 1 overexpressing animal;

[0033] (b) a step of administering a candidate substance to the animal;

[0034] (c) a step of performing a pain behavioral experiment on animals administered the above candidate substance and normal animals; and

[0035] (d) A step of selecting the candidate substance as a pain-relieving substance when the pain-responsive behavior of an animal administered the candidate substance is the same as the pain-responsive behavior of a normal animal.

[0036] As one embodiment of the present invention, the pain behavioral test of step (c) may be at least one selected from the group consisting of the Von Frey test, the Pin prick test, the Spiny seeds test, and the Pinned Mat Preference Test.

[0037] In addition, the present invention provides a pharmaceutical composition for preventing or alleviating pain, comprising as an active ingredient an agent that suppresses the expression or activity of Tentonin 1.

[0038] As one embodiment of the present invention, the agent that inhibits the activity of tentorin 1 may be NMB-1 (noxious mechanosensation blocker 1).

[0039] As another embodiment of the present invention, the agent that suppresses the expression of the tentonin 1 may be an oligonucleotide containing a sequence complementary to a part or all of a gene encoding tentonin 1, and specifically, may be an RNAi containing a sequence complementary to a part of a gene encoding tentonin 1, such as siRNA, miRNA, shRNA, etc., and may be a guide RNA.

[0040] The present invention identifies a novel function of tentonin 1 and provides it as a target for the treatment of acute and chronic pain. The present invention confirms that tentonin 1 acts as a mechanochannel to mediate slow adaptive currents induced by mechanical stimulation in sensory Schwann cells, and confirms an analgesic effect on various pain-inducing stimuli in the tentonin 1 knockout animal model. Therefore, the present invention is expected to be utilized as a platform for the development of analgesics that can be used for the treatment of neuropathic pain, etc.

[0041] Figure 1 is B6.Ttn1 -cre X RCL -ChR2 / EYFP This diagram confirms the expression of TTN1 in mouse smooth skin tissue. Thin fibers (red triangles) of YFP-expressing cells are stained with PGP9.5, and the cell bodies of these cells express Sox10 (arrows) (scale bar = 10 µm). [Abbreviations: TTN1; TTN1, PGP9.6; neurofilament marker; Sox10; sensory Schwann cell marker]

[0042]

[0043] Figure 2 (A) is a photograph of sensory Schwann cells stained with Sox10 antibody among dermal cells isolated from mouse foot skin, (B) shows the results of sorting Sox10-positive and -negative Schwann cells by FACS (Fluorescence Assisted Cell Sorter), (C) single cell RNA sequencing of cells sorted by FACS, and the results of unsupervised clustering analysis of Sox10 and S100b-positive cells among them show six clusters. (D) shows the results of confirming the expression level of mechanistic channel genes expressed by these cells [Abbreviations: Tmem150a; Tentonin 1, Tmem150b; Tentonin 2, Tmem150c; Tentonin 3, S10; Sox10, S100b; sensory cell markers]

[0044]

[0045] Figure 3 shows the results of analyzing the mechanical current characteristics of sensory Schwann cells:

[0046] (A) Representative trace of mechanical currents measured from a sensory Schwann cell in a littermate.

[0047] (B) Mean mechanocurrent measured in sensory Schwann cells cultured from littermate and TTN1 tissue-specific knockout (TTN1-cKO) mice. *, p < 0.05 (Student's T-test).

[0048] (C) Average inactivation time (τ1and τ2) values ​​at the measured mechanical current

[0049] (D) Classification of sensory Schwann cells cultured from littermate and tissue-specific knockout (TTN1-cKO-TdT) mice based on mechanocurrent kinetics. [Abbreviations: NR; no mechanocurrent response; SA; slow inactivation; IA; intermediate inactivation; RA; fast inactivation]

[0050] (E) Representative recording showing inhibition of sensory Schwann cell mechanocurrent by NMB-1.

[0051] (F) Mean mechanical current magnitude before and after NMB-1 treatment. *, p < 0.05, ***, p < 0.001 (One-way ANOVA, Tukey's post-hoc test)

[0052] (G) Mean mechanical current magnitude before and after Gd3+ treatment. **, p < 0.01 (One-way ANOVA, Tukey's post-hoc test)

[0053]

[0054] Figure 4 shows the results of analyzing the mechanical current characteristics of HEK293T cells overexpressing Tentonin 1:

[0055] (A) Mechanical current traces of HEK293T cells overexpressing TENS-1 and control cells following mechanical stimulation.

[0056] (B) Average mechanical current magnitude of HEK293T cells overexpressing TENS-1 and control (Mock) cells.

[0057] ***, p < 0.001 (Oneway ANOVA, Tukey's post-hoc test)

[0058] (C) Average mechanocurrent inactivation time of HEK293T cells overexpressing TENS-1 and control cells.

[0059]

[0060] Figure 5 confirms that the current induced by tenosynovium 1 is regulated by the mechanical current inhibitor NMB-1 (Drew et al., 2007):

[0061] (A) Mechanocurrent inhibition trace by NMB-1

[0062] (B) Changes in the magnitude of mechanical current before and after NMB-1 treatment. **, p < 0.01 (Student T-test).

[0063] (C) Mechanocurrent inhibition trace by Gd3+

[0064] (D) Changes in the magnitude of mechanical current before and after NMB-1 treatment. *, p < 0.05 (Student T-test).

[0065]

[0066] Figure 6 shows the results of measuring the degree of activity in the tibial nerve by mechanical stimulation:

[0067] (A) Schematic diagram of nerve action potential measurement

[0068] (B-C) Action potential traces (B) and analysis of the results (C) in von-Frey fibers from littermate and tendonin-1 cKO mouse nerves. *, p < 0.05, Two-way ANOVA (Tukey's post-hoc test).

[0069] (DE) Action potential traces (D) and analysis of results (E) by clipping. *, p < 0.05, Student's T-test.

[0070] (FG) Action potential traces by brush (F) and analysis of results (G). ns; not significant, Student's T-test.

[0071]

[0072] Figure 7 shows the results of comparing the pain levels of Littermate and Tentonin 1 cKO mice in various acute pain models: (A) von Frey test, *, p < 0.05, **, p < 0.01, ***, p < 0.001, Two-way ANOVA (Tukey's post-hoc test) (B) Pin prick test *, p < 0.05, Student's T-test (C) Hargreaves test ns; not significant, Student's T-test.

[0073]

[0074] Figure 8 shows the results of examining the pain level according to the knockout of tentinin 1 in a spiny seed ingestion model:

[0075] (A) Image of a mouse and seed exploring a thorny seed.

[0076] (B) Comparison of the proportion of times littermates and Tentonin 1 cKO mice grasped spiked seeds. ***, p < 0.001, Student's T-test.

[0077] (C) Comparison of the time spent holding seeds. ***, p < 0.001, Student's T-test.

[0078]

[0079] Figure 9 shows the results of a location preference experiment on spiked and flat mats:

[0080] (A) A mat with two materials

[0081] (B) Actual time spent and movement paths of Littermate and Tentonin 1 cKO mice on the entire mat (left: spiked mat, right: flat mat)

[0082] (C) Comparison of the ratio of time spent on the two types of mats. *, p < 0.05, Student's T-test.

[0083]

[0084] Figure 10 shows the results of examining the pain level according to the knockout of tenonin 1 in a chronic pain model:

[0085] (A) Pain level assessment 3 weeks after Spared Nerve Injury (SNI) surgery in littermate and Tentonin 1 cKO mice (nerve injury neuropathic pain model). * p < 0.05, Two-way ANOVA (Neuman-Keul's post-hoc test).

[0086] (B) Pain severity measured over a 2-week period in a paclitaxel-induced neuropathic pain model. *, p < 0.05, ***, p < 0.001, ****, p < 0.0001. Two-way ANOVA (Bonferroni's post-hoc test).

[0087]

[0088]

[0089] Figure 11 shows the results of comparing the pain levels of two mice in a carrageenan-induced inflammatory pain model:

[0090] (A) Comparison of pain levels by von-Frey fibers before and after carrageenan injection. **, p < 0.01, ***, p < 0.001, Two-way ANOVA (Bonferroni's post-hoc test).

[0091] (B) Comparison of pain levels by von-Frey fibers 4 hours after carrageenan injection. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001. Two-way ANOVA (Bonferroni's post-hoc test).

[0092] (C) Comparison of pain levels by von-Frey fibers 24 hours after carrageenan injection. *, p < 0.05, ***, p < 0.001, ****, p < 0.0001. Two-way ANOVA (Bonferroni's post-hoc test).

[0093]

[0094] Figure 12 shows the results of confirming the degree of analgesic effect by NMB-1:

[0095] (A) Comparison of pain levels by von-Frey fibers before plantar injection of NMB-1

[0096] (B) Comparison of pain levels by von-Frey fibers 30 minutes to 1 hour after plantar injection of NMB-1. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001. Two-way ANOVA (Bonferroni's post-hoc test).

[0097]

[0098] Figure 13 is a schematic diagram simply representing the structure of sensory Schwann cells in the dermis.

[0099] The present invention was completed through a preliminary study on the function of tentonin 1, the function of which has not yet been elucidated. The present inventors confirmed that tentonin 1 is intensively expressed in sensory Schwann cells. Sensory Schwann cells are a type of Schwann cell that surrounds nociceptors and have been found to be important cells in pain generation. When mechanical stimulation is applied to sensory Schwann cells, a slow adaptive current can be observed. The present inventors confirmed that the size of the slow adaptive current after mechanical stimulation was significantly reduced in tentonin 1 knockout sensory Schwann cells. In addition, when mechanical stimulation was applied to HEK cells produced to overexpress tentonin 1, the above-described slow adaptive current was confirmed. From the above, it was found that tentonin 1 is a mechanochannel activated by mechanical stimulation.

[0100] The present inventors have verified through various animal experiments that tentonin-1 is a mechanistic channel that mediates pain. Tentonin-1 knockout mice showed less pain response to various acute mechanical pain stimuli, such as the von Frey test and the pinprick test, compared to normal mice. In particular, the analgesic effect of tentonin-1 knockout was clearly evident in a test showing the response of freely moving mice to a thorn prick. From these results, the present inventors confirmed that tentonin-1 can be a target for a potent acute analgesic.

[0101] The present inventors confirmed the analgesic effect of the tentonin-1 knockout model not only in acute pain but also in chronic pain. Tentonin-1 knockout mice exhibited stronger analgesic effects than controls in chronic pain, including inflammatory pain induced by carrageenan injection, neuropathic pain induced by spinal nerve injury (SNI), and neuropathic pain induced by paclitaxel treatment.

[0102] From the above, it can be seen that an analgesic effect for acute and chronic pain can be obtained by inhibiting tentonin 1. Accordingly, the present invention provides tentonin 1 as a target for treating acute and / or chronic pain, and provides a platform for developing an analgesic for acute and / or chronic pain through screening for tentonin 1 inhibitors.

[0103]

[0104] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0105]

[0106] [Experimental Method]

[0107] 1. Mouse line

[0108] All mice were treated with the approval of the Korea Institute of Science and Technology (KIST). Mice were housed in individual cages with food and water provided on a 12-hour light-dark cycle. Mice aged 8-12 weeks were used. Sox10 iCreERT2 (abbreviated Sox10 Cre , stock number 027651), Rosa26R ChR2-EYFP (Ai32, stock number 012569), Rosa26R tdTomato (Ai9, stock number 007909) genetically modified mice were generated by Jackson Laboratory and Ttn1 fl / fl, Ttn1-P2A-Cre-P2A-dTomato (Ttn1Cre-dTomato) mice were generated by Macrogen. In addition, Ttn1 fl / fl ;Sox10 Cre / + (Ttn1 cKO), Ttn1 fl / fl ;CMV Cre / + (Ttn1 KO), and Ttn1 cre Mice (20-24 g) were used in the experiment.

[0109]

[0110] 2. Tamoxifen administration

[0111] Tamoxifen (Sigma) was dissolved in corn oil at a concentration of 20 mg / ml and injected intraperitoneally for 5 consecutive days. The injection dose was adjusted to approximately 75 mg of tamoxifen per kg of mouse, and the injection site was disinfected with 70% ethanol before injection.

[0112]

[0113] 3. Fluorescent tissue staining

[0114] Smooth skin and fur tissues were separated from the B6.Ttn1-cre-tdTomato X RCL-ChR2 / EYFP mouse line constructed above and fixed with 4% paraformaldehyde (PFA) at 4℃ for 4 hours. The fixed tissues were cryosectioned at a thickness of 20 μm and attached to slide glass. After washing twice for 5 minutes using PBS-0.05% Tween 20 solution (washing buffer), the tissues were reacted for 1 hour in a solution of Normal donkey serum (1:30) in PBS-0.5% Triton X-100 to perform permeabilization and blocking. After treating with primary antibody and reacting for more than 12 hours at 4℃, they were washed three times for 5 minutes each with washing buffer. After reacting with a secondary antibody conjugated to the desired fluorescent protein at room temperature for 2 hours, the cells were washed three times for 5 minutes each with washing buffer, stained with DAPI for nuclei, and mounted with a cover glass. The above tissues were imaged using a confocal microscope (LSM800, Zeiss).

[0115]

[0116] 4. Cell culture and transformation

[0117] HEK293T cell lines were cultured in DMEM / High Glucose (Gibco) containing 10% FBS and 100 U / ml penicilin / streptomycin. Transfected with 1 μg of the Ttn1-pIRES2-AcGFP1 plasmid using Fugene (Promega), and cultured for 48 h in a 5% CO2, 37°C incubator. Cells were seeded on poly-L-lysine-coated 35-mm culture dishes.

[0118]

[0119] 5. Primary culture method of sensory Schwann cells

[0120] Mice 10-14 days after birth were sacrificed with CO2, and the paws were rapidly separated in cold HBSS (TermoFisher Scientific) containing 100 U / ml penicillin and 100 μg / ml streptomycin. The plantar skin was then removed, and the skin tissue, from which nerve bundles and muscle tissues had been removed, was placed in cold HBSS containing 4 mg / ml collagenase / dispase (Sigma Aldrich) and incubated at 37°C for 30 minutes. Afterwards, the epidermal layer was removed, and the remaining dermis was cut into small pieces, placed in HBSS containing collagenase / elastase (Worthington) and incubated at 37°C for 30 minutes. The chopped tissues were mechanically stimulated using a 1 ml pipette coated with FBS to detach cells. The separated cell suspension was slowly filtered through a 40 μm cell strainer and centrifuged at 300 g for 6 minutes. The cell pellet was resuspended in Schwann cell medium (DMEM) supplemented with D-valine (Miclev, Cat. #AL251), 2 mM glutamine (ThermoFisher Scientific, Cat. #23030081), 10% FBS (Sigma, Cat. #2442), 1% N2 (Life Technologies, Cat. #17502001), 100 U / ml penicillin, 100 μg / ml streptomycin, 20 μg / ml bovine pituitary extract (Sigma, Cat. #P1476), 0.5 μM forskolin (Sigma), and 2 ng / mL heregulin-β1 (Sigma). Cells were seeded on coverslips coated with poly-L-lysine (Sigma) and laminin (Sigma) and incubated at room temperature for 30 min at 37°C.

[0121]

[0122] 6. Electrophysiological techniques

[0123] Whole-cell current recordings were performed using an Axopatch 200B amplifier (Molecular Devices). Whole-cell recordings were created by opening the plasma membrane using electrode tips. The glass electrode resistance was approximately 3 MΩ, the membrane voltage was held at -60 mV, and the junctional potential was adjusted to zero. The solutions used for patch clamping were as follows:

[0124] Intracellular solution: 130 mM CsCl, 2 mM MgCl1, 10 mM HEPES, pH of the solution is adjusted to 7.2 using CsOH.

[0125] Extracellular solution: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, pH of the solution is adjusted to 7.2 using NaOH.

[0126] The osmolarity of both solutions was adjusted to 290–300 mOsm using D-mannitol. Data were sampled at 5 kHz and filtered at 1 kHz using a Digidata 1440 (Molecular Devices). Curve fitting was performed using Clampfit 10.0 (Molecular Devices).

[0127] Mechanical stimulation was performed by stimulating the cell surface with a polished glass probe. Mechanical stimulation of sensory Schwann cells began approximately 1 μm from the cell surface and progressed in increments of 0.26 μm, from 1.3 to 7.7 μm. Each stimulus lasted 600 ms. The probe was controlled using a micromanipulator (NC5, Kleindiek Nanotechnik). Depending on the deactivation time constant (DT) of the mechanical current, it was classified as rapidly (RA), intermediately (IA), or slowly (SA) adapting. The following threshold values ​​were used: τ < 10 ms, 10 < τ < 30 ms, and τ > 30 ms.

[0128]

[0129] 7. Animal pain behavioral experiment

[0130] - The von Frey test was performed using a method from a previous study. Mice were placed in a plastic cylinder with a mesh floor and allowed to acclimate for 1 hour. After that, increasing von Frey filaments from 0.02 g to 4 g were applied vertically to the plantar surface of the hind paw until flexion occurred. The mouse's response to each filament was scored as follows: 0 (no response); 1 (movement of the mouse's toes without moving the leg); 2 (withdrawal); 3 (protection of the paw); 4 (licking or biting the stimulated paw). A nocifensive response rate of 3 or 4 in each trial was considered nocifensive behavior. The experimenter was blinded to the mouse genotype during all experiments.

[0131] The pin prick test was performed by pressing a sharp pin into the plantar surface of the hind paw of a mouse. The pin was applied until it passed through the mesh, a 1-cm length. A licking or biting response to the stimulus was considered nocifensive behavior. All experiments were conducted blinded to the mouse genotype.

[0132] - The thermal pain test involved placing mice on a glass plate, encasing them in a plastic cylinder, and allowing them to acclimate for one hour. The stimulus was delivered by applying a laser device beneath the glass plate to a constant intensity of heat. The time it took for the mice to exhibit an avoidance response to the thermal stimulus was measured to assess their thermal sensitivity.

[0133] - For the spiny seed test, mice were fasted for 18 hours. On the day of the experiment, they were transferred to a transparent plastic cylinder and allowed to acclimate for 15 minutes. They were then presented with spiny seeds coated with oligosaccharides. Two cameras were used to record mouse behavior within 15 minutes, analyzing the number of attempts to approach the seeds and the time spent grasping or biting them. Seeds were randomly selected to be as consistent in size and shape as possible, and littermates served as controls. Furthermore, the experimenter was blinded to the genotype of the mice during all experiments.

[0134]

[0135] 8. In vivo observation of tibial nerve fibers

[0136] Littermates or TTN1-KO mice (8–12 weeks old) were deeply anesthetized (using a 3:2 mixture of 50 μL Zoletil 100 (Virbac) and Rompun (Bayer) cocktail) and surgically exposed the tibial nerve of the right leg. The nerve was kept hydrated with saline. The nerve was transected at the proximal end before joining with other branches to form the sciatic nerve. A suction electrode with a 70 μm diameter tip was used to secure a tight junction between the nerve tip and the medial wall, and nerve activity was recorded. The success of the junction was confirmed by observing the nerve response to brush stimulation of the mouse's right paw. The electrode was coupled to an ISO-80 amplifier (World Precision Instruments), filtered at 0.3 and 1 kHz to remove low- and high-frequency interference, and digitized at a sampling rate of 10 kHz with a Digidata 1550B (Molecular Devices). For analysis, we used the threshold search mode within Clampfit's event detection function. The upper threshold was manually selected by observing the first 15 seconds of the trace, ensuring that the noise peak with the highest amplitude was below the threshold. Spikes with amplitudes higher than the threshold were counted as action potentials.

[0137]

[0138] [Experimental Results]

[0139] Example 1. Confirmation of Tentonin 1 expression in sensory Schwann cells present in the skin.

[0140] Because a tentonin-1 antibody has not been developed, it is difficult to confirm the expression of tentonin-1 by immunohistochemical staining. Therefore, a mouse line was established to confirm the expression pattern of tentonin-1. Specifically, EYFP (Enhanced Yellow Fluorescent Protein) expression was induced in tentonin-1-expressing cells by crossing RCL-ChR2 / EYFP mice, which induce Cre protein-dependent expression, with B6.Ttn1-cre-tdTomato mice.

[0141] Mouse paw skin was cut into 20-micrometer-thick sections and fluorescently stained for PGP9.5, a nerve marker, and Sox10, a sensory Schwann cell marker. The skin sections were stained with PGP9.5 and Sox10 antibodies, and then compared with the expression of EYFP, a reporter gene for tentorin 1, under a confocal microscope.

[0142] As shown in Figure 1, fibers expressing EYFP overlap with fibers stained with PGP9.5, and Sox10 staining also overlaps what appears to be the cell bodies of these cells. In particular, it was confirmed that EYFP-PGP9.5-expressing fibers extend deep into the dermal layer.

[0143] The above results indicate that sensory Schwann cells express tentonin 1. In particular, it can be seen that tentonin 1-expressing cells are distributed in the epidermal layer along the nerve endings.

[0144]

[0145] Example 2. Confirmation of expression of tensinin 1 in sensory Schwann cells.

[0146] In a different way from Example 1, we confirmed whether tentonin 1 is expressed in sensory Schwann cells. Schwann cells were isolated and cultured from the plantar skin of 10-day-old (P10) RCL-ChR2 / EYFP:Sox10-Cre mice. Schwann cells were relatively small, round, and had two or three protrusions (Fig. 2(A)). Sox10-positive and -negative cells were sorted using FACS (Fig. 2(B)). Among the cells sorted by FACS, Sox10-positive cells were subjected to single cell RNA sequencing, and unsupervised clustering analysis of Sox10 and S100b-positive cells was performed, and a total of six clusters were identified (Fig. 2(C)). Subsequently, the expression levels of mechanistic channel genes expressed by the cells were confirmed (Fig. 2(D)).

[0147]

[0148] Example 3. Confirmation of slow adaptation (SA) currents induced by mechanical stimulation in sensory Schwann cells.

[0149] Next, we performed electrophysiological experiments to determine how sensory Schwann cells respond to mechanical stimulation. Schwann cells were cultured primarily from mouse paw skin, and YFP-fluorescent sensory Schwann cells were identified among the cultured cells. Their current responses to mechanical stimulation were measured using the patch-clamp technique. Thin glass electrodes were placed on the sensory Schwann cells to create whole cells. Mechanical stimulation was applied to the cells, and the resulting currents were measured. When mechanical stimulation was applied to sensory Schwann cells, a current appeared immediately, which then rapidly and slowly decayed. This current, called the slowly adapting (SA) current, is nearly identical to the mechanocurrent elicited by tentonin 3 (Hong et al., 2016) (Fig. 3, A and D). However, in cells knocked out of tentonin 1, the SA current induced by mechanical stimulation was significantly reduced (Fig. 3, B). Additionally, the proportion of SA currents was significantly reduced in TEN-1 KO cells (Fig. 3D). These results suggest that SA-type mechanocurrents in sensory Schwann cells are primarily generated by TEN-1.

[0150] Mechanocurrents in nociceptive Schwann cells were inhibited by noxious mechanosensation blocker 1 (NMB-1), which is known to inhibit SA-type mechanocurrents in DRG neurons (Fig. 3, E and F). Mechanocurrents in nociceptive Schwann cells were also reversibly blocked by Gd3+, a mechanochannel-specific blocker (Fig. 3, G).

[0151]

[0152] Example 4. Confirmation of the mechanical channel properties of Tentonin 1.

[0153] Tentonin 1 is a homologue of tentonin 3, with 43.1% amino acid similarity (23.9% identity). To determine whether tentonin 1 can function as a mechanochannel like tentonin 3, mouse tentonin 1 was overexpressed in HEK293T cells. When mechanical stimulation was applied to HEK cells expressing tentonin 1, a SA current with typical SA-type current characteristics was generated (Fig. 4, A and B). The tentonin 1-induced current was observed to inactivate rapidly (τ1 = ~ 10 ms) and then slowly (τ2 = ~ 300 ms), which is similar to the SA mechanocurrent found in sensory Schwann cells (Fig. 4, C).

[0154]

[0155] Example 5. Confirmation that the tenocyte-1 mechanocurrent is inhibited by NMB-1.

[0156] We confirmed that the mechanocurrent observed after mechanical stimulation in cells overexpressing Tentonin 1 was suppressed by NMB-1 treatment (Fig. 5A and B). In addition, since Gd3+ nonspecifically inhibits mechanochannels, we confirmed that the SA current induced by mechanical stimulation was reduced by Gd3+ treatment in the cells overexpressing Tentonin 1 (Fig. 5C and D). This proves that Tentonin 1 is a mechanochannel, and in particular, an SA-type mechanochannel.

[0157]

[0158] Example 6. Confirmation of reduced cutaneous nociceptive nerve activity due to mechanical stimulation in Tentonin 1 KO mice.

[0159] First, we confirmed whether tentonin-1 actually modulates neural signals that detect mechanical stimulation in peripheral nerves. The tibial nerve was exposed by excising the skin of the left hindlimb of deeply anesthetized mice, and nerve activity was measured at the distal end using a suction electrode (Fig. 6A). Changes in the action potential of the tibial nerve were measured when various mechanical stimuli were applied to the sole. When the sole of a control mouse was mechanically stimulated with a von Frey filament, tibial nerve activity was observed, and this activity increased with increasing mechanical stimulation intensity (Fig. 6B and C). However, tibial nerve activity in tentonin-1 KO mice to the same stimulus was significantly reduced compared to the control group (Fig. 6B and C). Furthermore, when the sole of a control mouse was clamped with a clip, the tibial nerve showed strong activity. However, significantly less neural activity was observed in Tentonin-1 KO mice (Fig. 6D and E). In contrast, neural activity when a weak stimulus, such as brushing, was applied did not differ between the two groups (Fig. 6F and G). These results suggest that Tentonin-1 is a mechanochannel and molecular sensor that detects strong mechanical stimuli in the skin.

[0160]

[0161] Example 7. Confirmation of the analgesic effect of tetanyphin 1 knockout in an acute pain model.

[0162] To determine whether tentonin-1 mediates acute pain induced by mechanical stimulation, we performed an experiment to measure the severity of acute pain using tentonin-1 knockout (KO) mice. When the hind paws were stimulated with von Frey fibers of various diameters, control mice exhibited pain behaviors starting from a bending force of 0.16 g, and the frequency of pain behaviors increased significantly when fibers of 1 to 4 g were stimulated (Fig. 7A). In contrast, tentonin-1 knockout (KO) mice showed significantly less pain sensation (Fig. 7A). In other words, the pain threshold was also higher at 0.6 g than the control group, and although pain responses were observed even at high-intensity stimuli (1 to 4 g), they were significantly less severe than the control group (Fig. 7A). Similarly, in the pin prick test, tentonin-1 knockout (KO) mice experienced significantly less paw pain than the control group (Fig. 7B). However, in the case of the Hargreaves test, which measures pain caused by hot heat, there was no difference in the degree of pain behavior between the tentonin 1 KO mice and the control group (C in Figure 7).

[0163] Acute pain was assessed in a freely roaming animal model. When honey-coated seeds with thorns (Xanthium strumarium L) were placed in front of mice that had been food-restricted for more than 18 h, the mice attempted to grasp and eat the seeds (Fig. 8A). However, control mice experienced pain in their forepaws and could hardly lift or eat them. Surprisingly, tentonin-1 KO mice held the thorny seeds for extended periods of time and occasionally ingested them (Fig. 8B and C).

[0164] A place preference experiment was conducted to determine how long mice stayed on two types of mats: spiked and flat (Fig. 9A). Control mice stayed on the spiked mat approximately 25% longer than on the flat mat. However, Tentonin-1 cKO mice stayed significantly longer on the spiked mat than on the flat mat (Fig. 9B and C).

[0165] In summary, the above results show that mice with depleted tenonin-1 in their sensory Schwann cells experience significantly less mechanical pain caused by mechanical stimulation. This demonstrates that tenonin-1 acts as a molecular sensor that detects strong mechanical stimuli in sensory Schwann cells.

[0166]

[0167] Example 8. Confirmation of the analgesic effect of tetanyphin 1 knockout in a chronic pain model.

[0168] To determine whether tentonin-1 mediates pain in chronic pain models, we created several chronic pain models. When we created a mouse model of spinal nerve injury (SNI) and assessed the degree of pain sensitivity to mechanical stimulation, the control group showed a significant hypersensitivity to von Frey filament stimulation due to nerve injury (Figure 10A). In contrast, tentonin-1 KO mice showed no analgesic effect initially after SNI surgery but exhibited a significant analgesic effect from day 7 (Figure 10A). Similarly, tentonin-1 KO mice showed a more significant analgesic effect in a model of neuropathic pain induced by the anticancer drug paclitaxel (Figure 10B). Notably, tentonin-1 KO mice felt almost no pain, similar to the group that did not receive paclitaxel (Figure 10B).

[0169] We also investigated whether inflammatory pain induced by carrageenan is mediated by tendonin 1. To this end, carrageenan, which induces inflammation, was injected into the paw pad, and mechanical allodynia and hypersensitivity were observed in von Frey fibers 4 and 12 hours later, and compared with the pre-injection response. Littermates exhibited severe allodynia and hypersensitivity 4 hours after carrageenan injection (Fig. 11, B and C). However, allodynia and hypersensitivity were significantly reduced in tendonin 1 KO mice (Fig. 11, B and C).

[0170] In summary, the above results show that mice with depleted tenonin-1 in sensory Schwann cells experience significantly less pain in chronic neuropathic and inflammatory pain. This suggests that tenonin-1 plays a crucial role in mediating chronic mechanical pain.

[0171]

[0172] Example 9. Confirmation of the analgesic effect of treatment with NMB-1, a potent inhibitor of tetanyphin 1.

[0173] Previous animal experiments have shown that tentonin-1 is a key factor in the development of acute and chronic pain. This suggests that a substance that inhibits tentonin-1 will have analgesic effects. To demonstrate this, we injected NMB-1, a tentonin-1 inhibitor, into the hind paw of mice and tested the pain relief it produced. The analgesic effect was compared to the mechanical pain evoked by von-Frey fibers before and after NMB-1 injection. As shown in Figure 12, mice treated with NMB-1 showed a significantly less pain response than the control group to strong mechanical stimulation (>0.6 g). This suggests that compounds that inhibit tentonin-1 have analgesic effects. Therefore, this suggests that tentonin-1 may be a target for the development of new analgesics, and that compounds that inhibit tentonin-1 may be developed into new analgesics.

[0174] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0175] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

Claims

1. (1) Step of preparing cells overexpressing Tentonin 1 (Tentonin 1 / TMEM150a); (2) A step of treating the candidate substance to the above cells; (3) A step of applying mechanical stimulation to the above cells; (4) a step of measuring slow adaptation current in the above cell; and (5) A method for screening a tenonin 1 activity inhibitor, comprising: a step of selecting the candidate substance as a tenonin 1 activity inhibitor if the slow adaptation current measured in the cell is lower than the slow adaptation current of a cell that has not been treated with anything; 2. In paragraph 1, A screening method, wherein the cells of step (1) above are cells transformed with a vector for expressing tensinin 1. 3.(1) Step of preparing cells overexpressing Tentonin 1; (2) A step of treating the candidate substance to the above cells; (3) A step of applying mechanical stimulation to the above cells; (4) a step of measuring the activity level of tensinogen 1 used in the above cells; and (5) A method for screening an analgesic, comprising: a step of selecting the candidate substance as a pain relief substance when the activity of tentonin 1 in the above cells is lower than the activity of tentonin 1 in cells that have not been treated with anything; 4. In paragraph 3, The above step (4) is performed by measuring the slow adaptation current in the above cell, The above step (5) is a screening method for selecting the candidate substance as a pain relief substance when the slow adaptation current measured in the cell is lower than the slow adaptation current of a cell that has not been treated with anything.

5. In paragraph 3, A screening method, wherein the step (1) is performed by transforming a cell with a vector containing a gene encoding tensinin 1 and selecting transformed cells.

6. In paragraph 3, A screening method wherein the above analgesic is used for treating neuropathic pain.

7. Mechanical stimulus-refractory animal model with Tentonin 1 knockout or knockdown.

8. In paragraph 6, The above animal is a mouse, a mechanical stimulus refractory animal model.

9. As an animal model overexpressing Tentonin 1, The above animal model is an animal model that overreacts to mechanical stimulation.

10. In paragraph 9, The above animal is a mouse, an animal model. 11.(a) Step of preparing the animal model of Article 9; (b) a step of administering a candidate substance to the animal; (c) a step of performing a pain behavioral experiment on animals administered the above candidate substance and normal animals; and (d) A method for screening an analgesic, comprising: a step of selecting the candidate substance as a pain-relieving substance when the pain-responsive behavior in an animal administered the candidate substance is identical to the pain-responsive behavior in a normal animal.

12. In paragraph 11, A screening method, wherein the pain behavioral test of step (c) above is at least one selected from the group consisting of the Von Frey test, the pin prick test, the spiny seeds test, and the pinned mat preference test.

13. A pharmaceutical composition for preventing or alleviating pain, comprising as an active ingredient a preparation that suppresses the expression or activity of tendonin 1.

14. In paragraph 12, A pharmaceutical composition for pain relief, wherein the agent that inhibits the activity of tensinogen 1 is NMB-1 (noxious mechanosensation blocker 1).

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