WO2018147518A1 - Pharmaceutical composition for preventing or treating afferent disease comprising protein of tentonin 3 or polynucleotide encoding same as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating an afferent disease comprising a protein of Tentonin 3 or a polynucleotide encoding the same as an active ingredient. Specifically, when inhibiting Tentonin 3 (TTN3), a component of a mechanosensitive channel protein, it was confirmed that mechanosensitive current is reduced, an activity of fusimotor sensory nerves in mice is greatly reduced, and blood pressure and pulse rate of mice are greatly reduced. Therefore, a protein of Tentonin 3 or a polynucleotide encoding the same may be useful as a pharmaceutical composition for preventing or treating an afferent neurological disease and hypotension.

Description

Pharmaceutical composition for the prevention or treatment of afferent disease comprising the protein of Tentonin 3 or a polynucleotide encoding the same as an active ingredient

The present invention relates to a pharmaceutical composition comprising a protein of Tentonin 3 (Tentonin 3, TTN3) or a polynucleotide encoding the same as an active ingredient for the prevention or treatment of afferent disease.

Mechanosensation refers to external sensations and internal mechanosensory stimuli and is essential for the survival of animals. In mammals, physiological responses are required for tactile and tactile feelings, propriocption, and pain perception. Additionally, physiological functions such as hearing, body balance, baroreceptor reflexes, muscle activity of blood vessels, and blood volume control in the kidneys require the detection of mechanical sensory stimuli. Tactile stimulation is essential in everyday life such as perception or catching objects, walking, communicating with others, eating food, and even maternity care. Tactile stimuli are converted into nerve signals and transmitted to the spinal cord through major afferent nerves, Meissner corpuscles, Merkel disks, Ruffini endings, Pacsinian corpuscles, And specialized terminal organs in the skin, such as other terminal organs in the hair follicles of hair.

The mechanoreceptors of the skin are divided into low and high threshold mechanoreceptors, or fast and slow adapting receptors, which rely on their response to delayed mesensory stimuli. Thus, the morphology, skin location, and sensitivity or adaptation properties of the mechanoreceptors can exhibit a wide variety of responses to the individual tactile sensations.

Ion channels sensitive to tactile stimulation are known to mediate mechanically activated (MA) currents. The recently discovered mechanosensitive channel (mechanosensitive channel) (MscL) produces mechanoacceptive currents that function as osmotic valves in bacteria, while the DEG / ENaC and NOMPC families are the Caenorhabditis elegans and Drosophila melanogaster. It acts as a component of mechanotransformation channels in mechanosensory neurons.

These channels have not been found in mammals, but recently Piezo 1 and 2 (Piezo 1 and 2) have been newly discovered in mammals, and these channels are known to be opened by mechanical sensory stimuli and close immediately after being opened. They are expressed in dorsal-root ganglion (DRG) neurons and Merkel cells, and also rapid adaptation of currents in which the gene defects of Piezo2 respond to mechanistic stimulation in these cells. Piezo 2 is considered an RA type mechanical channel because it significantly reduces the MA current, which is important for tactile sensation, as it is known as an ion channel that responds to low threshold mechanical sensory stimuli important for skin tactile sensation. It is considered a channel. In addition, Piezo 1 is known to be involved in blood vessel formation.

However, DRG neurons respond to a variety of currents to mechanistic stimuli. They show a cationic current that deactivates rapidly, slowly, or moderately after activation, meaning that different mechanical channels are present. Among the DRG neurons, Piezo 2 is likely to have an ion channel that is distinct from Piezo 2, as it is involved in a mechanical current that rapidly deactivates (adapts) after activation.

On the other hand, proprioception (or proprioception) is a sense of the position or movement of muscles in the tertiary space, which is essential for the coordination of muscles essential for exercise, posture and fine muscle movement (Akay et al. , 2014; de Nooij et al., 2015; Proske and Gandevia, 2012). Intrinsic sensation requires sensory information from muscles, skin and joints, of which sensory signals from the muscle spindle (MS) are the most important. This muscle sensory nerve is very sensitive to the muscle elongation that occurs when the muscle contracts, and the mechanical channels present in the muscle sensory nerve detect this movement and send it to the central nervous system. Response is an essential signal for muscle control in our body.

 In addition, blood pressure control is an essential device for maintaining circulatory metabolism as a physical action for maintaining blood pressure at a desirable level, and is performed by pressure receptors present in nerves such as the carotid artery and the aorta. Blood pressure regulating nerves, including afferent fibers from these pressure receptors, play a role in regulating blood pressure, so the blood pressure control response of the baroreceptor is also essential for life support.

Accordingly, the present inventors endeavored to discover other mechanically activated (MA) channels in human cells in addition to Piezo 1 and Piezo 2, resulting in slow adapting, SA. ) Tentonin 3 (TTN3) channels were found, and when the mouse was inhibited, the coordination of muscles was inhibited and mean blood pressure and heart rate were significantly higher than those of normal mice. By confirming the lowering, the pharmaceutical composition comprising a protein of Tentonin 3 or a polynucleotide encoding the same as an active ingredient can be usefully used as a pharmaceutical composition for the prevention or treatment of afferent neurological disease and hypotension.

An object of the present invention is to provide a pharmaceutical composition comprising a protein of Tentonin 3 (TTN3) or a polynucleotide encoding the same as an active ingredient for the prevention or treatment of afferent neurological disease.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of afferent neurological disease comprising a protein of Tentonin 3 (TTN3) or a polynucleotide encoding the same as an active ingredient.

In addition, the present invention comprises the steps of: 1) treating the test substance to the cell expressing the Tentonin 3 gene; 2) measuring the expression level of Tentonin 3 gene in step 1); And 3) selecting a test substance in which the expression level of the tentonin 3 gene in step 2) is reduced compared to an untreated control group, and a method for screening a candidate substance for preventing or treating afferent neurological disease.

The present invention also provides a pharmaceutical composition for the prevention or treatment of hypotension, comprising a protein of Tentonin 3 or a polynucleotide encoding the same as an active ingredient.

The present invention also provides a method for preventing or treating afferent neurological disease, comprising administering to a subject a protein of Tentonin 3 or a polynucleotide encoding the same.

The present invention also provides the use of a protein of Tentonin 3 or a polynucleotide encoding it for use in the manufacture of a medicament for the prevention or treatment of afferent neurological disease.

The present invention also provides a method for preventing or treating hypotension, comprising administering to a subject a protein of Tentonin 3 or a polynucleotide encoding the same.

In addition, the present invention provides the use of a protein of Tentonin 3 or a polynucleotide encoding the same for use in the manufacture of a medicament for the prevention or treatment of hypotension.

Inhibition of Tentonin 3 in the present invention, by confirming that the mechanical sensory current is reduced, the activity of the near meso sensory nerves of the mouse is greatly reduced and the blood pressure and pulse of the mouse is greatly reduced, the protein of Tentonin 3 or encoding it Pharmaceutical compositions comprising a polynucleotide as an active ingredient can be usefully used as a pharmaceutical composition for the prevention or treatment of afferent neurological disease and hypotension.

1 is a diagram showing an estimated phase of TTN3 expected by the TMHMM program. Each dot represents the amino acid of mouse TTN3.

FIG. 2 shows a Phylogenetic tree of the TTN3 gene family between different species of phylum Chordata . Sequence alignments and phylogenies were shown using the CLUSTALW program. Cm, Chelonia mydas; Dr, Danio reiro ; Fc, Felis catus ; Gg , Gallus gallus ; Hs , Homo sapiens; Mm, Mus musculus ; Rn, rattus norvegicus; Xt , Xenopus tropicalis .

Figure 3 is a diagram showing the mRNA profile of the TTN3 gene. Quantitative PCR was performed from 21 adult mouse organs. Expression levels were normalized to mRNA levels of GAPDH.

4 is a diagram showing an anti-sense hybridization site (in situ hybridization) in DRG neurons using the (left, brown spots) and sense (right) strand. After in situ hybridization, cross sections were immunostained with neurofilament M (NFM) antibodies (pink).

FIG. 5 is a diagram showing the ratio of TTN3-positive neurons and the ratio of neurofilament M (NFM +), a marker of TTN3-positive neurons, among the total DRG neurons.

FIG. 6 is a diagram showing that current is activated by mechanistic stimulation in Ttn3 − and Gfp − transduced HEK293T cells, inducing MA current with slow inactive kinetics. After forming the whole cell, the mechanical sensory stimulus was applied for 600 ms, and the voltage was held at -60 mV.

FIG. 7 is a diagram showing currents activated by mechanosensory stimulation in HEK293T cells into which Ttn1 − and Ttn2 − were introduced . After forming the whole cell, the mechanical sensory stimulus was applied for 600 ms, and the voltage was held at -60 mV.

8 is Ttn3 - and Gfp - in HeLa cells by introducing a trait is a diagram showing the current activated by the mechanical sensory stimulation. After forming the whole cell, the mechanical sensory stimulus was applied for 600 ms, and the voltage was held at -60 mV.

9 is a diagram showing the average peak amplitude of MA currents activated by mechanical sensory stimulation in HEK293T and HeLa cells incorporating Ttn1- , Ttn2- , Ttn3- , and Gfp -types. Bars represent SEM and in parentheses the number of cells tested.

FIG. 10 is a diagram showing the ratio between the peak (I _MAX ) and the steady state (I _SS ) of the current measured in F11, HEK293T, and HeLa cells. The steady state current was measured at 600 ms.

11 is Ttn3 -, Gfp -, and siRNA (Scrmble) is an MA and the current measured in the F11 cell differentiation by introducing a trait, showing a degree of X tonin 3 (TTN3) gene expression in these cells.

12 is Ttn3 -, Gfp -, and siRNA (Scrmble) is also introduced into the transformant showing the MA current measured at the undifferentiated cells to F11.

13 is a siRNA (Scrmble), Ttn3 - a diagram showing the Ttn3 and average peak amplitude of the current activated by the machine MA sensory stimulus in introducing -siRNA transfected cells to differentiated F11.

FIG. 14 is a diagram showing the result of leveling the MA current I with the maximum peak current I _MAX activated by displacement of the mechanical sensory stimulus. Mechanical sensory stimulation was applied for 600 ms and displacement represents the distance of the mechanical sensory stimulus from the cell surface. Leveled currents were optimized with the Boltzmann equation given by I / Imax (%) = [(1 + exp (-(DD _1/2 ) / k))-1]. Wherein D represents the past (indentation) distance D _1/2 means a half of the maximum travel distance, and, k is the reaction sensitivity to trace station (indentation). Red dots (Red dots) represents the average value of D _1/2 s.

FIG. 15 is a diagram showing that knockdown of TTN3 suppresses SA MA currents in DRG neurons, showing one type of MA currents in DRG neurons into which siRNA (Scrmble) and Ttn3 -siRNA traits are introduced. DRG nerves were stimulated by mesensory stimulation that increased by 0.42 μm for 100 ms.

FIG. 16 is a diagram showing another type of MA current in DRG neurons into which siRNA (Scrmble) and Ttn3 -siRNA traits are introduced.

FIG. 17 shows another type of MA current in DRG neurons into which siRNA (Scrmble) and Ttn3 -siRNA traits are introduced. DRG nerves were stimulated by mesosensory stimulation increased by 0.84 μm for 600 ms.

FIG. 18 shows gene expression levels of TTN3 in DRG neurons transfected with siRNA (Scrmble) and Ttn3- siRNA (28 PCR cycles, 48 hours after transduction).

19 is a diagram showing peak currents of DRG nerves activated by 6.3 μm mesensory stimulation after transduction of siRNA (Scrmble) or Ttn3- siRNA. DRG neurons were classified as inactive kinetics of MA currents, and the inactivity time constants (τ i) for RA, IA, and SA MA currents were <10 ms, 10 <τ i <30 ms, and> 30 ms, respectively.

20 is a diagram showing a cell type histogram of DRG neurons transfected with siRNA (Scrmble) or Ttn3- siRNA.

FIG. 21 shows that TTN3 follows the biophysical characteristics of SA MA currents in DRG neurons. FIG. 21 shows that steady state currents are maintained during mesenchymal stimulation in F11 cells transduced with Ttn3 . The figure shows that it is maintained up to 1000 ms. The asterisk indicates the stationary current at the end of the mechanical sensory stimulus.

FIG. 22 shows MA currents in TTN3 expressing cells activated by mesensory stimulation with two rates of 18 and 66 μm / s.

FIG. 23 is a diagram showing the effect of conditioning stimulation on MA current of cells transduced with Ttn3 . FIG. A mechanical sensory stimulus with an increase of 0.42 μm and indentations of 2.9-4.6 μm for 100 ms was applied and a test pulse with a stimulus of 4.6 μm was performed.

FIG. 24 is a diagram showing current in peak and steady state induced by conditioning and test stimuli. FIG. It was leveling the current (I) of peaks with a maximum peak or stationary (I _MAX) or a stationary current (I _SS) (normalized).

25 is a diagram showing MA currents recorded in HeLa cells transduced with Ttn3 .

FIG. 26 is a diagram showing current-voltage (IV) correlation of steady state MA currents in TTN3 expressing F11 cells, measured in pipette solution conditions with different compositions. A voltage ramp (-80 to +80 mV) was applied during 600 ms, 4.2 μm mechanical sensory stimulation. The pipette solution contained 150 mM CsCl and the bath solution contained 150 mM NaCl, 150 mM KCl, 100 mM CaCl ₂ or 100 mM MgCl ₂ .

Fig. 27 is a diagram showing the ion permeability ratio (P _X / P _Cs ) of MA current in the steady state of F11 cells expressing TTN3.

FIG. 28 is a diagram illustrating a pharmaceutical profile of MA current involving TTN3, showing TTN3 involvement-MA current suppressed by 30 μM of gadolinium (Gd ³⁺ ) in F11 cells. FIG. The mechanical sensory stimuli were repeated and each inhibitor was treated for 1 minute before the second mechanical sensory stimulus and washed for recovery currents.

FIG. 29 shows TTN3 involvement-MA current inhibited by 30 μM FM1-43 in F11 cells. FIG. The mechanical sensory stimuli were repeated and each inhibitor was treated for 1 minute before the second mechanical sensory stimulus and washed for recovery currents.

30 shows TTN3 involvement-MA currents inhibited by 2.5 μM GsMTx4 in F11 cells. The mechanical sensory stimuli were repeated and each inhibitor was treated for 1 minute before the second mechanical sensory stimulus and washed for recovery currents.

31 is TTN3 involved - a diagram showing the reaction correlation-Gd ^{3 +} Gd ^{+ 3} concentration of inhibiting the MA current _{(IC 50 = 3.9 μM, n} = 7 - 8).

FIG. 32 shows TTN3 involvement-50 μΜ chloropromazine (CPZ), 10 μΜ HC-030031, 1 mM amyloide, 30 μΜ ruthenium red (RR), 30 μΜ on MA current Figure showing the effect of gadolinium (gadolinium, Gd), 30 μM FM1-43 and 2.5 μM GsMTx4.

Fig. 33 shows the position of TTN3 on the plasma membrane of HEK (HEK293T) and F11 cells transfected with pEGFP-N1 (STOP) -TTN1 or pEGFP-N1. Cells were stained with Hoechst33342.

34 is a plot showing that the inactivity ratio of the TTM3 MA currents fits well with a mono- or bi-exponential curve:

a: diagram showing TTM3 MA currents in F11 cells showing that the ratio of inactivation is in good agreement with the mono-exponential curve;

b: A diagram showing the distribution of inactivation time constant (τ i) of TTN3 expressed in HEK293T and F11 cells. The parenthesis shows the number of cells tested;

c: Figure showing TTM3 MA currents in F11 cells showing that the ratio of inactivation is well aligned with the bi-exponential curve; And

d: A diagram showing the distribution of TTN3 inactive time constants (τ1 and τ2) in HEK293T, HeLa, and F11 cells.

35 shows that TTN3 is abundantly located in the central spinal region of the extensor digitorum longus (EDL) of the hind limb muscles of normal (WT) mice, and the activity of renal-induced neurodischarge in TTN3 knockout mice. Decreased sensitivity to and muscle height:

a: photograph of the near spine of the extensor digitorum longus (EDL) of the hind limb muscle of the mouse;

b: Figure showing the distribution of TTN3 and neurofilament M in the central region of the proximal spine;

c: TTN3 and neuro- at annulospiral structures

Figure showing the distribution of filament M;

d: diagram showing activity of muscle kidney-induced neurodischarge; And

e: diagram showing afferent activity in the proximal spine observed in the first half of the elongation range (1-3 mm).

36 is a diagram showing that the motor coordination ability of TTN3 knockout mice is decreased through experiments such as hanging upside down, cross-section test, gait analysis, etc .:

a: inverted screen test;

b: beam walking test;

c: average speed;

d: swing speed;

e: stride length;

f: regularity index; And

g: phase dispersion.

37 is a diagram showing the decrease in blood pressure and pulse rate of TTN3 knockout mice by measuring the mean blood pressure and heart rate (heart rate) of TTN3 knockout mice:

a: blood pressure measurement results in TTN3 knockout mice compared to control (normal) mice; And

b: Heart rate measurement results in TTN3 knockout mice compared to control (normal) mice.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prophylaxis or treatment of afferent neurological disease comprising a protein of Tentonin 3 or a polynucleotide encoding the same as an active ingredient.

The tentonin 3 protein is composed of the amino acid sequence of SEQ ID NO: 9, characterized in that activated by mechanical sensory stimulation.

The protein may be a variant of an amino acid having a different sequence by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function of the protein. Amino acid exchange in proteins or peptides that do not alter the activity of the molecule as a whole is known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, or farnesylation. Accordingly, the present invention may include a polypeptide having an amino acid sequence substantially identical to a polypeptide having an amino acid sequence described by any one or more sequences selected from the group consisting of SEQ ID NO: 9, and variants or fragments thereof. Such substantially identical polypeptides may have homology with at least 80%, in particular at least 90%, more specifically at least 95%, with the polypeptides of the invention.

In a specific embodiment of the present invention, the inventors confirmed the phase, phylogenetic phylogeny and tissue distribution of Tentonin 3 and found that they have 96% and 99% amino acid sequence similarity of human and rat orthologs, respectively. (See FIG. 2), epididymis, pancreas, DRG, eye, brain, and spinal cord were found to show high expression (see FIG. 3). In situ hybridization analysis and distribution in mouse tissues confirmed that about one-third of the neurons in DRG were positive for tentonin 3 (see FIG. 4). It was confirmed that the co-movement, such as neuronal filament M (neurofilament M) containing (see Figure 5).

In addition, the present inventors measured the activity of Tentonin 3 in 293T cells, confirming that a strong current is activated (see FIG. 6), and the steady state current at 600 ms mesenchymal stimulation is TTN3 / HEK293T. It was confirmed that the cell was about 52.5% of the peak current (see FIG. 10). In addition, as a result of measuring the activity of Tentonin 3 by mechanical sensory stimulation in transduced F11 cells, it was confirmed that inducing MA current (see FIGS. 11 and 13), Tentonin 3 in DRG cells derived from mouse As a result of measuring what type of current is essential, it was confirmed that it is essential for a slow adapting current (see FIG. 19). In addition, it was confirmed results confirm the biophysical (Biophysical) characteristics of the X tonin 3, that the cation channel with high transmission for the Ca ^{2 +,} PCl / Because PNa is 0.13 ± 0.03 (n = 8) , Cl - relatively with respect to the It was confirmed that it has a high permeability (see Fig. 27). And as a result of confirming the pharmaceutical profile of the MA current using the channel blockers of Tentonin 3, it was confirmed that significantly inhibit the MA current (see Figs. 28, 29, 30 and 32).

In addition, the afferent nerves are the body cavity and visceral afferents, visual afferent, olfaction, auditor afferents, corticohypothalamic afferents. It is preferably one or more selected from the group consisting of hippocampohypothalamic fibers, amygdalohypothalamic fibers, amygdalohypothalamic fibers, hypothalamic fibers (Thalamohypothalamic fibers) and sheathed fibers (Tegmental fibers).

In addition, the hypothalamus, a centrally regulated physiological function, receives neuronal input from various motor, sensory and brain regions of the afferents to the hypothalamus. Root spindles, which are known to do, are essential for the control of intrinsic sensation, which senses the position and movement of muscles by detecting changes in the length of muscles, and thus is involved in motor coordination or fine movement. This intrinsic sensation function begins with the activity of the root sensory nerves in the proximal spine. When the muscle contracts or relaxes, the root nerve senses the change in muscle length and sends it to the central nervous system. Therefore, the mechanical channels in the near-sensory nerves are also essential for intrinsic sensory function.

In a specific embodiment of the present invention, the present inventors confirmed that Tentonin 3 is expressed in a large amount in the muscle sensory nerves (see FIG. 35A), and TTN3 knockout mice have a greater inhibition of the activity of the muscle sensory nerves than the control group. (See FIGS. 35D and e). In addition, as a result of observing the exercise control ability of TTN3 knockout mice, it was confirmed that the exercise control ability of the muscle is lower than that of the control group (see FIG. 36).

The pharmaceutical composition comprising the protein of Tentonin 3 of the present invention or a polynucleotide encoding the same as an active ingredient, preferably includes 0.0001 to 50% by weight of the active ingredient based on the total weight of the composition.

The pharmaceutical composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as needed. And other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets, such as aqueous solutions, suspensions, emulsions, and the like, which will act specifically on target organs. Target organ-specific antibodies or other ligands can be used in combination with the carrier so as to be able to be used. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

In addition, the present invention

1) treating the test substance to cells expressing the tentonin 3 gene;

2) measuring the expression level of Tentonin 3 gene in step 1); And

3) A method for screening a candidate substance for preventing or treating afferent neurological disease, comprising selecting a test substance in which the expression level of the tentonin 3 gene in step 2) is reduced compared to an untreated control group.

The cells are any of the group consisting of HeLa, HEK293T, F11, human DRG, retinal pigment epithelium (RPE), human keratocytes (HaCaT), hepatocellular carcinoma (HepG2) and human uterine carcinoma (SiHa) cells. One or more may be selected, but is not limited thereto.

The present invention also provides a pharmaceutical composition for the prevention or treatment of hypotension, comprising a protein of Tentonin 3 or a polynucleotide encoding the same as an active ingredient.

Blood pressure control is a blood pressure measuring sensor called a baroreceptor in carotid sinus, aorta, atrium, etc., which receives signals and controls the activity of sympathetic and parasympathetic nerves in the blood pressure control center. The same machine channel is likely to work.

In a specific embodiment of the present invention, the inventors confirmed that tentonin 3 (TTN3) knockout mice showed much lower mean arterial pressure and heart rate than the control group (see FIG. 37). ).

The present invention also provides a method for preventing or treating afferent neurological disease, comprising administering to a subject a protein of Tentonin 3 or a polynucleotide encoding the same. The subject may be a mammal, and in particular, may be a rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey, human, and the like.

Proteins or polynucleotides used in the present invention may be prepared for the purpose of oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal and the like.

Preferably, the protein or polynucleotide is used in injectable form. Thus, in particular, the area to be treated may be mixed with any pharmaceutically acceptable vehicle for injectable compositions for direct infusion. The protein or polynucleotide of the present invention may comprise a lyophilized composition which enables the composition of an injectable solution, in particular upon the addition of isotonic sterile solutions or dry especially sterile water or suitable physiological saline. Direct injection of proteins or polynucleotides into a patient's tumor is advantageous because it allows the treatment efficiency to be focused on the infected tissue. The dosage of the protein or polynucleotide used can be adjusted by various parameters, in particular by the gene, the vector, the mode of administration used, the disease in question or alternatively the duration of treatment required. In addition, the range varies depending on the weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of the patient. The daily dose is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once to several times a day.

The protein or polynucleotide may be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or It can be administered by intrathoracic injection and can be used in the form of a general pharmaceutical formulation.

The present invention also provides the use of a protein of Tentonin 3 or a polynucleotide encoding it for use in the manufacture of a medicament for the prevention or treatment of afferent neurological disease.

The present invention also provides a method for preventing or treating hypotension, comprising administering to a subject a protein of Tentonin 3 or a polynucleotide encoding the same.

In addition, the present invention provides the use of a protein of Tentonin 3 or a polynucleotide encoding the same for use in the manufacture of a medicament for the prevention or treatment of hypotension.

Thus, the present invention confirms that Tentonin 3 (TTN3) is activated by mesensory stimulation with slow inactive kinetics, is expressed directly in mice, and reduces mesensitivity currents when Tentonin 3 is inhibited by blockers. In addition, by confirming that the activity of the muscle sensory nerves, the muscle control capacity and the blood pressure are lowered in the tentonin 3 deficient mouse, the protein of the present invention or the polynucleotide encoding the same is afferent neurological disease. And it was confirmed that it can be usefully used as a pharmaceutical composition for the prevention or treatment of hypotension.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1 Molecular Cloning Step of the TTN Family

The following experiments were performed to construct fragments of the TTN gene family ( Ttn 1, 2, and 3 ).

Specifically, primers were designed using the mouse cDNA sequences of Ttn1 , 2, and 3 from NCBI databases (NM_144916.3, NM_177887, and NM_182841). 816, 717, and 750 bp of the respective Nucleotide fragments of Ttn1 , 2 and 3 were amplified from the kidney, small intestine, and epididymis cDNA libraries of adult C57BL / 6J mice. The primer sequences used below are shown.

1) Ttn1 forward (5 ′ ctcgaggccaccatgaccgcct 3 ′; SEQ ID NO: 1) and Ttn1 reverse (5 ′ aagcttgatcatggcaatactctcgggag 3 ′; SEQ ID NO: 2) such as restriction sites XhoI and HindIII;

2) Ttn2 forward (5′ctcgaggccaccatgtggaattacct 3 ′; SEQ ID NO: 3) and Ttn2 reverse (5 ′ aagcttgagcacctgtgccatctgta 3 ′; SEQ ID NO: 4) such as restriction sites XhoI and KpnI; And

3) Ttn3 forward (5'ctcgaggccaccatggacgggaagaaat 3 '; SEQ ID NO: 5) and Ttn3 reverse (5' ggtaccctcacctgatccgtctgatattc 3 '; SEQ ID NO: 6) such as restriction sites XhoI and Kpn .

It was cloned the Ttn 1, 2 and 3 fragment in pEGFP-N1.

For electrophysiological recordings, the vector was further mutated to include a stop codon between the gene and the GFP sequence (pEGFP-N1 STOP), and the gene was pIRES2-AcGFP1 to avoid GFP fusion effects. Sub-cloned.

The protein sequence of TTN 1

And MTAWILLPVSLSAFSITGIWTVYAMAVMNRHVCPVENWSYNESCSPDPAEQGGPKSCCTLDDVPLISKCGTYPPESCLFSLIGNMGAVMVALICLLRYGQLLEQSRHSWINTTALITGCTNAAGLVVVGNFQVDHAKSLHYIGTGVAFTAGLLFVCLHCVLFYHGATTPLDMAMAYLRSVLAVIAFITLVLSGVFFLHESSQLQHGAALCEWVFVLDILIFYGTFSYEFGTISSDTLVAALQPAPGRACKSSGSSSTSTHLNCAPESIAMI (SEQ ID NO: 7),

The protein sequence of TTN 2 is

MWNYLSLLPVILFLWAIAGIWIVFAIAVVNGSVDLNEGFPFISICGSYAPQSCIFGQVLNIGAALTVWICIVRHHQLRDWGVKTWQNQLILWSGILCALGTSIVGNFQDKNQKPTHLAGAFLAFILGNLYFWLQFFLSWWVKGLPQPGPHWIKSLRLSLCSLSTILIVAMIVLHALHMRSASAICEWVVAMLLFMLFGFFAVDFSILRGCTLHLHPRLDSSLPQAPSGSPNIQMAQVL and (SEQ ID NO: 8),

The protein sequence of TTN3 is

MDGKKCSVWMFLPLVFTLFTSAGLWIVYFIAVEDDKILPLNSAARKSGAKHAPYISFAGDDPPASCVFSQVMNMAAFLALVVAVLRFIQLKPKVLNPWLNISGLAALCLASFGMTLLGNFQLTNDEEIHNVGTSLTFGFGTLTCWIQAALTLKVNIKNEGRRAGIPRVILSAVITLCVVLYFILMAQDIHMYAARVQWGLVMCFLAYFGTLAVEFRHYRYEIVCSEYQENFLSFSESLSEASEYQTDQV is (SEQ ID NO: 9).

Example 2 Cell Culture and Transfection

In order to transduce the vector and siRNA prepared in <Example 1> was performed the following experiment.

Specifically, HeLa, HEK293T, and F11 cells were Dulbecco's modified supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and 100 U / ml penicillin / streptomycin. Cultured in Eagle (DMEM) medium. Cells were aliquoted onto glass coverslips in a 35 mm dish.

F11 cells were treated with 10 μM forskolin (Sigma) to induce differentiation into DRG-like cells. Forskolin-treated F11 cells were laminin-coated with DMEM supplemented with 1% FBS, 2 mM L-glutamine, and 100 U / ml penicillin / streptomycin in a 35 mm dish. (2 mg / ml) was dispensed on glass coverslips. Half of the medium was changed every two days.

Lipofectamine 2000 with pIRES2-AcGFP1-TTN1, pIRES2-AcGFP1-TTN2, pIRES2-AcGFP1-TTN3, pEGFP-N1 (STOP) -TTN1, pEGFP-N1 (STOP) -TTN2 or pEGFP-N1 (STOP) -TTN3 vectors (Life Technologies) or FuGene (Promega) were transduced for 48 hours.

Ttn3 And scrambled siRNA sets were designed, synthesized, and tagged with Cy3 fluorescence (Bioneer, Seoul, Korea). The siRNA was located at Nucleotide 421-439 and the sequence was Tnt3_siRNA sense 5'-GCUUGUGGAUAGUGUACUU (dTdT) -3 '(SEQ ID NO: 10) and antisense 5'-CGAACTCCTATCACATGAA (dTdT) -3' (SEQ ID NO: 11) to be. Cy3-tagged siRNAs were transfected into cultured DRG cells using Lipofectamine 2000 and siRNA-treated cells were incubated for 48 to 72 hours before use.

Example 3 Real-Time Quantitative PCR

All tissues were isolated from C57BL / 6J wild type mice and total RNA samples were extracted using Total RNA Extraction Kit (Intron). First strand cDNA was synthesized using the GoScript reverse transcription kit (Promega). Templates were amplified using Power SYBR Green PCR Master Mix (Applied Biosystems) and duplicated using ABI StepOne Plus and StepOne real-time systems. Product calibration and normalization were performed using a two-cycle threshold (Ct) method. Here, Ct represents (Ct (target gene)-Ct (reference gene)).

For mRNA expression analysis in other tissues, GAPDH was used as a reference gene and the following primers were used.

Ttn1 : 5'-ggtcctacaatgaatcctgctc-3 '(forward) (SEQ ID NO: 12),

5'-ggccaccataacagcaccc-3 '(reverse) (SEQ ID NO: 13);

Ttn2 : 5'-tgtcctggtgggtgaaagg-3 '(forward) (SEQ ID NO: 14),

5'-tcatagccacgatgaggatgg-3 '(reverse) (SEQ ID NO: 15);

Ttn3 : 5'-cgtgtggatgtttctacctctc-3 '(forward direction) (SEQ ID NO: 16),

5'-gcacccgatttccttgcag-3 '(reverse) (SEQ ID NO: 17); And

GAPDH: 5'-aggtcggtgtgaacggatttg-3 '(forward) (SEQ ID NO: 18),

5'-tgtagaccatgtagttgaggtca-3 '(reverse) (SEQ ID NO: 19).

Example 4 Electrophysiology

Whole-cell currents were recorded using a voltage-clamp method of patch clamp. After the giga seals were formed, the plasma membrane was broken under a glass pipet to form a whole-cell. The resistance of the glass pipette electrode was 2-3 MPa. The junction potential was adjusted to zero and holding voltage at -60 mV. Current was amplified with Axopatch200B (Molecular Devices), filtered at 0.5 kHz, digitized at a sampling rate of 5 kHz with Digidata 1440 (Molecular Devices), and stored on a computer. The stored current was analyzed using pClamp software (version 10.0, Molecular Devices).

Pipette solutions for recording current in cells included 130 mM CsCl, 2 mM MgCl ₂ , 10 mM Hips (HEPES), 2 mM Mg-ATP, 0.2 mM Na-GTP, and 25 mM D-mannitol with pH of CsOH Using 7.2. For DRG neuron recording, the pipette solution included 130 mM KCl, 2 mM MgCl ₂ , 5 mM EGTA, 10 mM Hips, 2 mM Mg-ATP, 0.2 mM Na-GTP and 20 mM D-mannitol. pH was adjusted to 7.2 with KOH. The bath solution included 130 mM NaCl, 5 mM KCl, 1 mM CaCl ₂ , 2 mM MgCl ₂ , 10 mM Hips and 10 mM D-mannitol at pH 7.2 adjusted with NaOH.

For ion selectivity, the pipette solution included 150 mM CsCl and 10 mM hippies at pH 7.2 adjusted with CsOH. The bath solution included 150 mM NaCl, 150 mM KCl, 150 mM NMDG-Cl, 100 mM CaCl ₂ or 100 mM MgCl ₂ and 10 mM Hips. pH was adjusted to 7.2 using NaOH, KOH or CsOH for divalent cation solutions. Osmolarity of all solutions was adjusted to 290 mOsm by the addition of D-mannitol.

To determine the current-voltage (IV) correlation, a voltage ramp from -80 to +80 mV was applied for 150 ms during the 600 ms mechanistic stimulation in the holcell. For block experiments, 50 μM chloropromazine (Sigma), 10 μM HC-030031 (Sigma), 1 mM amyloide (Sigma), 30 μM ruthenium red red) (Sigma), 30 μM FM1-43 (Biotium), 2.5 μM GsMTx-4 (Tocris) or 30 μM GdCl ₃ (Sigma) were added to the bath solution. Gd ^{3 +} dose for the inhibition - to obtain a response curve (dose-responsive curve), the standardized current (I / I _MAX) and applied to a heel formula (Hill equation).

I / I _MAX (%) = [1 + (K _D / [Gd ³⁺ ]) ⁿ ] ^-1

The K _D refers to half of the maximum effect of Gd ^{+ 3,} and n is the Hill coefficient means (Hill coefficient).

< Experimental Example  1> TTN3  Identify topology, phylogenetic phylogenetic tree, and tissue distribution

<1-1> TTN3 phase, phylogenetic tree check

In order to determine the phase, phylogenetic phylogenetic tree, and tissue distribution of TTN3, the following experiment was performed.

Specifically, for phylogenetic analysis, multiple sequence alignments and phylogenetic phylogenies were performed using the CLUSTALW program. GenBank accession IDs are shown in the table below.

Protein Acc. Gene Organism NP_001073975.1 Ttn3 H. sapiens human NP_878261.1 Ttn3 M. musculus mouse NP_001101824.1 Ttn3 R. norvegicus Rat XP_420558.4 Ttn3 G. gallus Chicken XP_005155606.1 Ttn3 D. rerio Zebrafish XP_002936503.1 Ttn3 X. tropicalis Frog XP_007065696.1 Ttn3 C. mydas Turtle XP_003985286.1 Ttn3 F. catus Cat NP_001026908.1 Ttn1 H. sapiens human NP_659165.1 Ttn1 M. musculus mouse NP_620807.1 Ttn1 R. norvegicus Rat XP_421633.3 Ttn1 G. gallus chicken NP_001038875.1 Ttn1 D. rerio Zebrafish NP_001016894.1 Ttn1 X. tropicalis frog XP_007069920.1 Ttn1 C.mydas turtle XP_003984259.1 Ttn1 F. catus cat NP_001268940.1 Ttn2 H. sapiens human NP_001136264.1 Ttn2 M. musculus mouse NP_001100946.1 Ttn2 R. norvegicus Rat NP_001032458.1 Ttn2 D. rerio Zebrafish NP_001015997.1 Ttn2 X. tropicalis frog XP_003997471.1 Ttn2 F. catus cat

As a result, the topology of TTN3 was predicted to have six transmembrane domains with short N- and C-terminus (FIG. 1), presumably the reentry region was located between transmembrane domains 1 and 2. appear. The Ttn gene was not found in the lower phyla of animals or plants (FIG. 2). TTN3 appears to be less associated with 26.5% and 28.1% sequence identity with the other two paralogs, TTN1 and TTN2, whereas TTN3, on the other hand, is responsible for 96% and 99% of human and rat orthologs, respectively. Had amino acid sequence similarity. Quantitative PCR analysis of three Ttn genes in 21 mouse tissues showed different expression patterns for each gene (FIG. 3), with TTN3 in epididymis, pancreas, DRG, eye, brain and spinal cord. It was confirmed to exhibit high expression.

<1-2> In situ hybridization analysis and tissue distribution of TTN3

In situ hybridization analysis and tissue distribution of TTN3 were performed as follows.

Specifically, the following experiments were performed for in situ hybridization analysis and immunofluorescence analysis. Adult mouse DRG was dissected and immediately frozen on isopentane. They were embedded in the optimal cleavage temperature compound and serially sectioned into 14 μm sections. The sequence of the Ttn3 mRNA target probe was constructed in Advanced Cell Diagnostics (Hayward, Calif.), And the probe targeting the bacterial gene dapB was used as a control.

In situ hybridization analysis of Ttn3 mRNA was performed on samples that were section frozen using the RNAscope 2.0 HD Detection Kit (Brown). Tissue sections were dried at 50 ° C. for 30 minutes and then fixed with 4% paraformaldehyde for 15 minutes. Tissue sections were progressively dehydrated with 50%, 70% and 100% ethanol for 5 minutes and pretreated with a probe kit before hybridization. After hybridization with Ttn3 mRNA at 40 ° C. for 2 hours, the RNAscope 2.0 HD detection kit was used for signal amplification. Chromogenic detection was performed using diaminobenzidine, and immunohistochemistry was used to detect primary antibodies against neurofilament M (NFM, 1: 200, Santa Cruz). Was performed using. Alexa 546-conjugated donkey anti-rabbit IgG was treated for 30 minutes (1: 500) and sections were washed after three washes and mounted.

As a result, it was confirmed that about 1/3 (849 / 2,690 neurons) were positive for TTN3 in DRG (FIG. 4). In addition, it was confirmed that about 36% of TTN3-containing neurons (302/849 neurons) co-migrated with neurofilament M, a marker of myelinated neuron (FIG. 5).

Experimental Example 2 MA Channel Screening and Gene Identification

In order to find MA channels other than Piezo1 and 2, bioinformation surveys were conducted.

Specifically, human DRG cells and six human cell lines, human embryonic kidney (HEK) 293T, HeLa, retinal pigment epithelium (RPE), human keratocytes (keranocyte, HaCaT), hepatocellular carcinoma (HepG2), and DNA chips (Agilent) were screened after hybridization with mRNA isolated from human uterine carcinoma (SiHa) cells. Slowly adapting MA channels were found mainly in DRG neurons, so we selected six cell line genes whose mating intensity was less than 70% of the DRG cell strength.

Additionally, genes with known potential or genes encoding fewer than five transmembrane domains were selected and narrowed down to six genes, Gdpd2, Sidt1 , Tmem26 , Tmem150c , Tmem163 and Tmem229a . Candidate genes selected from DRG neurons isolated from adult mice were transfected with Cy3 tagged siRNAs and tested for a decrease in SA current in response to mesensory stimulation.

As a result, it was confirmed that the knockdown of one gene, TMEM150C, showed a significant decrease in the amplitude of the SA current (FIG. 19).

Experimental Example 3 Activity by Mechanical Stimulation

<3-1> Determination of TTN3 activity in transduced 293T cells

Tentonin 3 (TTN3) is a relatively small protein with 6 transmembrane domains and 249 amino acids. Tentonin 3 was overexpressed in HEK293T cells was found to be targeted to the plasma membrane (Fig. 33).

In order to test the mechanosensitivity of Tentonin 3, the following experiment was performed.

Specifically, holcell current (whole) using the technique used in Example 4 from various cell lines transduced with pIRES2-AcGFP1-TTN3 or pEGFP-N1 (STOP) -TTN3 as in <Example 2>. -cell currents) were recorded. The plasma membrane surface of TTN3 expressing cells was stabbed with a piezo-electrically driven glass probe. A fire-polished electrode (tip diameter: 3-4 μm) was placed at an angle of about 50 ° to the cell surface, and the probe was placed on a micromanipulator (Nano-controller NC4, Kleindiek Nanotechnik, Germany). Probe movement is controlled by a joystick or computer. The initial position of the probe on the cell surface is determined by observing in the marks on the cell surface through a microscope (x400). A retraction is then made, which is considered to be the initial point of all mesensory stimuli, from which the mesensory stimulus increases from 2.5-7.2 to 0.42 μm, moving forward in the glass probe. The typical time for mechanistic stimulation is 100 or 600 ms.

As a result, when mechanical sensory stimulation was applied to the cells as shown in FIG. 6, a strong current was activated (FIG. 6). After fast activation, the current exhibited rapid inactivation and again slow inactive MA current, as long as the mechanistic stimulus was maintained for a long time. The residual current lasted for a few seconds when the mechanical sensory stimulus was removed and was not activated (FIG. 6). Of the 66 tested cells, 34 cells (52%) showed MA currents greater than 300 pA.

30 Gfp - the transduced cells, only seven were in response to mechanical stimulation with sensory small current amplitudes (amplitudes) (Figs. 6 and 9). In addition, two homologs, TTN 1 and TTN 2, showed very low intensity MA currents when expressed in HEK293T cells (FIGS. 7 and 9). The ratio of inactivation of MA currents in TTN3-expressing cells fits well with the mono- or bi-exponential curves (FIG. 34). The inactivation time constant of constant of mono-exponential curve (τi) of the single-exponential curve was 286.1 ± 41.1 ms (n = 7). In TTN3 / HEK293T cells, the steady state current during 600 ms mechanistic stimulation was about 52.5% of the peak current (FIG. 6). Due to the slow inactivity time constant (> 30 ms), the TTN3-dependent MA currents were grouped with the SA currents observed in the DRG neurons.

In addition, large MA currents with inactive kinetics with slow mechanistic stimulation were induced in HeLa cells transduced with Ttn3 . The rate of inactivity best fits the bi-exponential curve. The time constants for fast (τ1) and slow inactivity (τ2) of the bi-exponential curve were 12.0 ± 1.6 and 213.0 ± 17.1 ms (n = 19), respectively. In addition, a portion of the control cells after mesensitizing stimulation induced a small current (127.7 ± 15.5 pA, n = 22) (Figs. 8 and 9).

<3-2> Measurement of TTN3 Activity in Transduced F11 Cells

In order to measure the activation by mechanistic stimulation in F11 cells transduced with Ttn3 , the following experiment was performed.

Specifically, F11 is a hybridized cell line between mouse neuroblastoma, N18TG-2 and rat DRG neurons. F11 cells can be differentiated into DRG-like neurons by treatment with forskolin (10 μΜ, 4-6 days).

As a result, it was confirmed that mesensory stimulation on differentiated F11 cells transduced with Ttn3 induced MA current (2,397 ± 229 pA, n = 25) with slow inactive kinetics (FIGS. 11 and 13). Mesensory stimulation on F11 cells transfected with scrambled siRNA induced small but strong MA currents (187.9 ± 22.2 pA, n = 19) (FIG. 11). This seems to be due to the inclusion of a small amount of endogenous TTN3 (endogenous TTN3). When mouse Ttn3 -siRNA was transduced into F11 cells, MA currents were significantly inhibited (60.6 ± 9.0 pA, n = 14, p <0.001, Student's t-test) and were not treated with forskolin When Ttn3 was transduced in F11 cells, it was confirmed that large MA currents were activated by mechanistic stimulation (FIGS. 12 and 13).

When various mesensory stimuli were applied to the surface of F11 cells, MA currents were observed which could be broken down by grade (FIG. 14). Half the maximum distance for TTN3 to be activated is 3.5 ± 0.1 μm (n = 6), which is equivalent to that of human Piezo1 (4.7 ± 0.3 μm, p <0.01, Student's t-test) expressed in F11 cells. Significantly lower than the figures. Therefore, it was confirmed through experiments that TTN3 is activated by mechanistic stimulation when heterologously expressed in various cell types.

Experimental Example 4 Essentiality of TTN3 for Slow Adaptive MA Current in DRG Neurons

The following experiments were conducted to find out what type of current TTN3 is essential for.

Specifically, culture of DRG cells was prepared to isolate DRG neurons. DRG nerves of the thoracic and lumbar spine were dissected from 7-8 week old mice and obtained in cold medium (DMEM / F12, Life Technologies). The isolated DRG was incubated in a medium containing 2 mg / ml collagenase IA (Sigma) at 37 ° C. for 45 minutes. Cells are washed three times with Hank's balanced salt solution (HBSS, Life Technologies) without Ca ^{2 +} -and Mg ^{2 +} -, washed two or three times with culture medium, suspended and round glass Triturated with 1 ml pipette prior to dispensing on a Fisherslip. DMEM / F12, 10% FBS, 50 ng / ml Neurogrowth (Life Technologies), 5 ng / ml glial cell line-derived neuropathy in 95% air / 5% CO ₂ atmosphere for 48-72 hours before use of cells The culture was carried out in a culture medium containing a neurotrophic factor (Life Technologies) and 100 U / ml penicillin / streptomycin.

Mechanical sensory stimulation on isolated DRG neurons is rapidly adapting (RA) and intermediatel adapting with three distinct types of currents, τi of <10 ms, 10 <τi <30 ms and> 30 ms, respectively. , IA) and SA (FIGS. 15, 16 and 17).

Three types of MA currents are distinguished by inactive kinetics present in DRG neurons, where kinetically defined DRG neurons respond differently to delayed tactile stimuli. RA neurons tend to be induced in short phase discharges, while SA neurons tend to induce delayed discharges. SA neurons tend to follow mechanistic stimuli at high frequencies, and the frequency of action potentials in SA neurons is proportional to the intensity of mechanistic stimuli. The degree of current change in RA neurons in response to changes in the rate of mechano-stimulation is much greater than in SA neurons. Thus, RA neurons are more suited to responsible for dynamic changes in mechanistic stimulation, while SA neurons are more suitable for responsible for changes in identity.

When transfected with scrambled siRNA, the proportion of DRG neurons with RA, IA, and SA MA currents was relatively small in the group of DRG neurons (20-25 μm diameter, 206 cells), 47%, 20%, respectively. And 20%. In contrast, when Cytn- tagged Ttn3 siRNA was transduced into the DRG neuron, the proportion of cells with SA MA current was reduced, whereas the proportion of cells with RA and IA kinetics was unaffected ( 20). Similarly, Ttn3- siRNA transduction induced a significant decrease in the amplitude of SA currents but not RA or IA currents (FIG. 19). Thus, it was confirmed that TTN3 is essential for SA current in DRG neurons.

Experimental Example 5 Confirmation of Biophysical Characteristics of TTN3

In order to determine the biophysical characteristics of TTN3, an experiment as shown in Example 4 was performed.

As a result, the steady state current of TTN3 induced by the mechanesthetic stimulus was maintained for up to 1,000 ms while the mechanistic stimulus lasted for a long time (FIG. 21). In addition, the intensity of the MA TTN3 current was proportional to the speed of the mechanical sensory stimulus, and the faster the mechanical sensory stimulus was applied, the greater the current was observed (FIG. 22). As a two-stage mechanical sensory stimulation protocol, first, a 4.6-μm test step was performed following a 100 ms conditioning step with 0.42 μm increments and indentations of 2.9 to 4.6 μm. Although the intensity of the control step varied, the MA current at the peak or steady state of the test step did not change (FIGS. 23 and 24). Thus, much of the MA current continued in the test phase. In addition, a sinusoidal step was applied after a sinusoidal MA current followed by a 200-ms conditioning step (FIG. 25). This result indicates that some TTN3 is still active after persistent mechanistic stimulation that is characteristic of SA currents found in DRG neurons.

The MA TTN3 current is a cation with a reverse potential of 6.7 ± 0.7 mV (n = 9) with 150 mM CsCl and 150 mM NaCl in pipette and bath solutions, respectively, and the bath solution is 150 mM Given the reversal potential when changed to NaCl, 150 mM KCl, 100 mM CaCl ₂ , or 100 mM MgCl ₂ , the permeability ratio between Na +, K +, Mg ^{2 +} or Ca ^{2 +} and Cs ⁺ ( The permeability ratio (Px / Pcs) was 1.31 ± 0.04 (n = 9), 1.24 ± 0.05 (n = 6), 1.41 ± 0.15 (n = 6) and 2.01 ± 0.11 (n = 8), respectively (Figure 27). Thus, it was confirmed that the TTN3 cation channel with a high transmission for the Ca ^{2 +.} In addition, since P _Cl / P _Na is 0.13 ± 0.03 (n = 8), it was confirmed that TTN3 has a relatively high permeability to Cl ⁻ .

Experimental Example 6 Pharmacology of MA Current

In order to determine the pharmaceutical profile of the MA current, the following experiment was performed.

Specifically, in the DRG neurons isolated from the <Experiment 3> is the current MA dolrini were blocked with Titanium (gadolinium, Gd ^{+ 3)} and other blockers (blocker). As in <Example 2>, F11 cells transduced with Ttn3 were stimulated with three consecutive mesensory stimuli (3.6 or 4.2 μm) at the same distance.

As a result, it was confirmed that MA TTN3 current is blocked by gadolinium (Gd ³⁺ , 30 μM), a known mechanical sensory channel blocker. Gadolinium significantly inhibited MA currents in TTN3 expressing cells (FIGS. 28 and 32). Half of the maximum concentration for inhibition (IC ₅₀ ) was 3.9 μM (n = 7-8) (FIG. 31). In addition, TTN3 current was found to be markedly reduced by ruthenium red (30 μM) and FM1-43, a fluorescent dye known to block MA currents in DRG neurons (FIGS. 29 and 32). ). In addition, purified tarantula toxin (GsMTx4, 2.5 μM), known to inhibit MA currents in various cell types, was found to strongly inhibit MA TTN3 currents (FIGS. 30 and 32). MA TTN3 currents were not blocked by chloropromazine (50 μM), a membrane modifier known to inhibit MA TREK1 channels, and HC030031 (10 μM), a TRPA1 blocker known to inhibit mechanistic sensory currents. Amiloride (1 mM), known to block the ENaC current, was found to slightly reduce the MA current.

Experimental Example 7 Expression of TTN3 in the Proximal Vertebrae

To determine whether TTN3 is expressed in the proximal spine, the extensor digitorum longus (EDL) of 7-8 week old mice was isolated, cut to 10 μm, and fixed with 4% polyformaldehyde. After washing with PBS, the cells were blocked with 4% bovine serum albumin for 1 hour and treated with Polyclonal TTN3 (1: 700) or NFM (sc-51683, SantaCruz, 1: 500) at 4 ° C for 48 hours. After washing again, Alexa Fluor 546-conjugated goat anti-rabbit (A11010, Life Technology, 1: 500) and Alexa Fluor 488-conjugated goat anti-guinea pig (AP193F, Invitrogen, 1: 800) were treated, and the nucleus was Hoechst 33342. (H3570, Thermofisher Scientific, 1: 2000) was stained for 10 minutes and observed using a confocal microscope (LSM700, Carl Zeiss).

As a result, it was confirmed that TTN3 is abundantly located in the roots of the extensor digitorum longus (EDL) of the hind muscles of the mouse (FIG. 35A). TTN3 was prominently observed in the central region of the root spine, where the nuclei of the intravenusal muscles were gathered, and surrounded by the intravenusal muscles responsible for the typical primary afferent in the roots. It was located with neurofilament M at annulospiral structures (Figs. 35b and c). However, TTN3 was not observed in the roots of mice knocked out of TTN3.

Since the presence of TTN3 in the near spine was confirmed, the function of TTN3 in the aortic centripetal activity was examined through the control (Wild Type, WT) and TTN3 knockout mice.

In order to perform extracellular neural recording in the peroneal nerve connected to the separated extensor Digitorum Longus muscle (EDL), the mouse is anesthetized with isoflurane, and then the extensor Digitorum Longus muscle, EDL) connected to the peroneal nerve 128 mM NaCl, 1.9 mM KCl, 1.2 mM KH ₂ PO ⁴ , 26 mM NaHCO ₃ , 0.85 mM CaCl ₂ , 6.5 mM MgSO ₄ , and 10 mM glucose (pH 7.4) ) Were separated from the solution at 4 ° C., and the long toe flat muscle was connected to the sensor with a thin silk suture to evaluate muscle strength and control ability. The sensor's output signal was amplified to determine the size of the muscle extension associated with the force applied to the muscle. A glass suction electrode having a tip diameter of 70 μm was prepared, filled with a bath solution, and the free end of the peroneal nerve was suctioned to the electrode end. In order to determine the apical centripetality, the aspiration was repeated until the activity of the nerve cells was observed during the extension of the muscle. The kidney was repeated three times of 2 mm in length and 5 seconds in length. Two more times. The electrode signal in the tip was amplified with World Precision Instruments (ISO-80), digitized at a sampling rate of 10 kHz using Digidata 1322 (Molecular Devices) fmf, and filtered at 1 kHz. And stored on your computer. The stored current was analyzed using pClamp software (version 10.0, Molecular Devices) fmf, and the spikes beyond one standard deviation of noise were calculated by identifying the action potential of the nerve.

As a result, 5 seconds of 2 mm elongation (~ 10 mm) in WT in the control (WT) caused barrage of nerve discharges, but in the TTN3 knockout mice It was found that the activity of muscle stretch-evoked discharge in afferents was dramatically reduced (FIGS. 35 d and e). Decreased afferent activity in the proximal spine was observed throughout the muscle range (1-3 mm) (p <0.0001, two-way ANOVA, F (1,132) = 56.5), and TTN3 knockout mice The sensitivity of the kidney (sensitivity, slope) is significantly reduced compared to the control (WT) mouse (5.81 ± 0.72 vs 2.77 ± 0.36 spikes / mm, p <0.001, n = 23-24, Student -t test) (FIG. 35E). These results show that TTN3 is involved in kidney-evoked near-central afferent activity.

Experimental Example 8 Motor Control Loss in TTN3 Inhibited Mice

Because sensory input of the muscles is essential for muscle coordination, experiments are conducted to hold the grid to the limbs (arms and legs) of the mouse to test muscle control and muscle strength. It was.

The mouse was prepared to grasp using a limb a welded wire mesh (560 mm x 510 mm) consisting of 11.5 mm x 11.5 mm (0.8 mm diameter), which was slowly turned upside down and smoothed It was installed to be fixed to the upper 30cm. Time was measured until the mouse dropped.

As a result, TTN3 knockout mice had shorter hanging times compared to normal mice (WT). Control (WT) mice hung for more than 10 minutes but TTN3 knockout mice mostly did not hang for more than 2 minutes (FIG. 36 a).

In addition, a beam walking test was performed, in which a mouse was trained to cross three times a day for two days in a plastic average beam having a width of 12 mm (length 80 cm and height 50 cm), and then a narrower 6 mm plastic average beam. The experiment was performed three times. After crossing the balance beams, the mice were allowed to hide in dark spaces, and the movements were captured in video and analyzed.

As a result, TTN3 knockout mice showed more than three times the slip of limbs (arms and legs) compared to the normal group, and the time required to cross the average was significantly increased (4.6 sec vs 6.5 sec, p <0.01, n = 10-11, Student-t test) (FIG. 36 b).

And to analyze the gait of the mice was performed as follows.

The gait analysis of the mouse was performed using CatWalk system (Noldus Information Technology), and the experimental device was composed of 1.3m long glass layer where the light illuminates the floor in the dark space. deflection phenomenon is used to shine brightly. Experiments were taken by video and saved to computer. Mice were allowed to run three consecutive runs, and mice were considered successful if they walked for more than 5 seconds. The gait footings of each mouse were identified and labeled and analyzed using Catwalk XT software, and the average of three test values was used. Swing is the speed at which the limb moves when the mouse moves, and stride is the length of the gait cycle. In addition, the regularity index refers to the ratio of the normal foot pattern to the total number of foot footprints of the mouse, and phase dispersion refers to the target footing based on the fixed step length of another foot. Mean initial contact ratio.

As a result, TTN3 knockout mice showed abnormal results compared to the control. Gait in TTN3 knockout mice was found to significantly slow the swing and stride length compared to the normal group (WT) (FIGS. 36 c, d and e). And TTN3 knockout mice showed a decrease in fronto-hind limb coordination (Figs. 36 f and g).

In contrast, inhibition of TTN3 does not appear to affect general motor activity as well as tactile or painfulness, indicating that TTN3 is involved in motor coordination through the near afferent centripetal nerve in mice. do.

Experimental Example 9 Blood Pressure and Pulse Measurement in TTN3 Inhibited Mice

Next, the following experiment was performed to determine the change in blood pressure and pulse rate in TTN3 knockout mice.

Specifically, blood pressure of the TTN3 knockout mouse and the control (normal) mouse was performed by a non-invasive blood pressure measurement method, and a CODA 8-Channel high throughput non-invasive blood pressure system of Kent Scientific was used. This is similar to a human blood pressure monitor, and a cuff was placed on the tail of the mouse, and the air pressure was applied thereto, and the air pressure was measured by slowly lowering the air pressure. First, keep the temperature of the platform constant at 33-35 ℃, fix the mouse in the restrainer, and then fix the occlusion cuff (O-cuff) and volume pressure recording cuff (VPR- cuff). At this time, the mice were habitated for at least 5 minutes and then blood pressure was measured. Blood pressure was automatically measured by a sensor attached to the VPR, and the blood pressure was measured 20 times for each mouse, and the average value was viewed as blood pressure. At the same time, the pulse rate (heart rate) was automatically recorded and the average value was viewed as the pulse rate.

As a result, as shown in FIG. 37, blood pressure was significantly lower in TTN3 knockout mice compared to the control (normal) mice. There was no statistical significance due to the small number of experimental animals (FIG. 37 a) (p <0.09. N = 3). In addition, as a result of measuring the heart rate, a significant decrease in the pulse rate was confirmed in TTN3 knockout mice (FIG. 37B) (p <0.04, N = 3).

Claims (11)

Pharmaceutical composition for the prevention or treatment of afferent neurological disease comprising a protein of Tentonin 3 or a polynucleotide encoding the same as an active ingredient. According to claim 1, Tentonin 3 protein is a pharmaceutical composition for the prevention or treatment of afferent neurological disease, characterized in that consisting of the amino acid sequence of SEQ ID NO: 9. The method of claim 1, wherein the tentonin 3 is a pharmaceutical composition for the prevention or treatment of afferent neurological disease, characterized in that activated by the mechanical sensory stimulation. The method of claim 1, wherein the afferent nerves are the body cavity and visceral afferents, visual afferent, olfactory, auditory afferents, cortical hypothalamus (Corticohypothalamic afferents), Hippocampohypothalamic fibers, Amygdalohypothalamic fibers, Thalammohypothalamic fibers, and Tegmental fibers. Pharmaceutical composition for the prevention or treatment of afferent neurological disease. 1) treating the test substance to cells expressing the tentonin 3 gene; 2) measuring the expression level of Tentonin 3 gene in step 1); And 3) A method for screening a candidate substance for preventing or treating afferent neurological disease, comprising selecting a test substance in which the expression level of the tentonin 3 gene in step 2) is reduced compared to an untreated control group. The method of claim 5, wherein the cells are HeLa, HEK293T, F11, human DRG, retinal pigment epithelium (RPE), human keratocytes (keranocyte, HaCaT), hepatocellular carcinoma (HepG2) and human uterine carcinoma (SiHa) A method for screening a candidate agent for the prophylaxis or treatment of afferent neurological disease, characterized in that it is any one selected from the group consisting of cells. A pharmaceutical composition for preventing or treating hypotension, comprising a protein of Tentonin 3 or a polynucleotide encoding the same as an active ingredient. A method for preventing or treating afferent neurological disease, comprising administering to a subject a protein of Tentonin 3 or a polynucleotide encoding the same. Use of a protein of Tentonin 3 or a polynucleotide encoding the same for use in the manufacture of a medicament for the prevention or treatment of afferent neurological disease. A method of preventing or treating hypotension, comprising administering a protein of tentonin 3 or a polynucleotide encoding the same. Use of a protein of tentonin 3 or a polynucleotide encoding the same for use in the manufacture of a medicament for the prevention or treatment of hypotension.

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