WO2004064861A1

WO2004064861A1 - Nerve cell death inhibitor

Info

Publication number: WO2004064861A1
Application number: PCT/JP2004/000548
Authority: WO
Inventors: Hiroshi Ueda
Original assignee: Nippon Chemiphar Co., Ltd.
Priority date: 2003-01-24
Filing date: 2004-01-22
Publication date: 2004-08-05
Also published as: JP4564922B2; JPWO2004064861A1

Abstract

It is intended to provide a nerve cell death inhibitor containing, as the active ingredient, prothymosin α1 or a protein or a peptide having a function equivalent thereto to thereby establish a means of treating diseases associated with nerve cell death.

Description

Description Nerve cell death inhibitor Technical field

The present invention relates to a nerve cell death inhibitor comprising prothymosin a1 (hereinafter referred to as "PTA1") as an active ingredient and a therapeutic agent for a disease associated with nerve cell death. Background art

In the human brain, tens of billions of neurons form a complex network, but if the mechanism of neurogenesis from a few neural stem cells is not taken into account, the number of cells will basically be It just decreases. The brain uses various protective mechanisms against various exogenous and endogenous stresses for a long life span of several hundred years, and maintains its survival. The brain's own protective mechanisms are influenced by neuro-glia and neuro-neural communities, and work to maintain their advanced role. The most well-known mechanisms of neuroprotection are those that are functioned by molecules such as neurotrophic factors and cytoplasm. These neurotrophic factors are known to have the function of suppressing programmed neuronal death (apoptosis) seen under various stress conditions. Another mechanism is neurogenesis, and recent reports have reported that neurogenesis is enhanced during cerebral ischemia stress, but it is expected that it is not sufficient to supplement the nerves that undergo massive cell death. You. During cerebral ischemia, necrosis, which is destructive cell death, is observed in the central part of the ischemic core, but this cell death dissipates the cell contents to the outside. It should spread to the surroundings. However, a few days later, a phenomenon specific to apoptosis, such as fragmentation and condensation of cells and phagocytosis by microglia, is observed in a surrounding area called penambula. Apoptosis seen in Penampra is considered to function as a kind of protective mechanism that prevents injury to the whole brain by localizing the injury site (see Non-Patent Document 1). Sometimes the form of cell death from necrosis to apoptosis The conversion is presumed to be caused by a particular substance (see Non-Patent Document 1), but to date no such substance has been identified.

The present invention has been made under the above-mentioned technical background, and an object of the present invention is to specify a substance capable of converting a cell death form from necrosis to apoptosis.

[Non-Patent Document 1] Hiroshi Ueda, Wakako Hamanabe Journal of Pharmaceutical Sciences of Japan, 119, 79-88 (2002)

The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, PTA1, which was considered to be a precursor of thymosin, acts on (A) nerve cells to suppress necrosis. Necrosis inhibitory function), (B) Acts on neurons and promotes apoptosis (pro-apoptosis function), and (C) Acts on microglia to promote secretion of substances (neurotrophic factors) that inhibit apoptosis It has been found that it has a function to indirectly suppress apoptosis (indirect apoptosis suppression function). From the above functions, when acting only on nerve cells (such as when PTA1 is added to cultured neurons), PTA1 converts the cell death form of neurons from necrosis to apoptosis. PTA1 inhibits both neuronal necrosis and apoptosis when it acts on and microglia (such as when administering PTA1 in vivo).

The present invention has been completed based on the above findings.

That is, the present invention is a nerve cell death inhibitor comprising PTA1 or a protein or peptide having a function equivalent thereto as an active ingredient.

Further, the present invention is a therapeutic agent for a disease associated with nerve cell death, comprising PTA1 or a protein or a peptide having a function equivalent thereto as an active ingredient.

Further, the present invention relates to a peptide represented by the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a peptide substantially identical to these peptides.

Hereinafter, the present invention will be described in detail.

Although PTA1 is a known protein, it has been reported by the present inventors that PTA1 has the above-mentioned necrosis-suppressing function, apoptosis-promoting function, and indirect apoptosis-suppressing function. It was discovered for the first time.

These three functions are presumed to be expressed by the following mechanism (Fig. 1).

(A) Necrosis suppression function-

1) Binding of PTA1 to receptors in nerve cells

2) Transfer of glucose transport to cell surface

3) Increase in intracellular glucose

4) Increase in ATP production from mitochondria

5) Suppression of necrosis

(B) Apoptosis promoting function

1) Binding of PTA1 to receptors in nerve cells

2) Increased expression of Bax and Bim proteins and decreased expression of Bel-2 and Bd-xL proteins in neurons

3) Release of cytochrome c from mitochondria

4) Caspase activation

5) Activation of CAD

6) Promotion of application

(C) Indirect suppression of apoptosis

1) Binding of PTA1 to glial cells-

2) Increased expression of neurotrophic factor and cytokine genes in glial cells

3) Activation of neurotrophic factor receptor (Trk) and cytokine receptor in nerve cells

4) Control of appointment

From the above findings, PTA1 can be used as a nerve cell death inhibitor. PTA1 used for the neuronal cell inhibitor is not particularly limited, and PTA1 derived from human, PTA1 derived from rat, PTA1 derived from mouse, and the like can be used. The amino acid sequence of PTA1 actually obtained from these organisms is shown in FIG. The amino acid sequences of three types of PTA1 are shown in SEQ ID NO: 1 (human), SEQ ID NO: 2 (rat), and SEQ ID NO: 3 (mouse). Figure 3 shows the aligned figures. PTA1 derived from organisms other than human, rat, and mouse includes PTA1 and PTA1 derived from force can also be used. These amino acid sequences of PTA1 are registered in GenBank and the like as Accession No. TNB0A1 and CAC39397, respectively.

Instead of PTA1, a protein or peptide having a function equivalent to that of PTM can be used. Here, "a protein or a peptide having a function equivalent to PTA1" refers to a protein or a protein having all or a part of the necrosis-suppressing function, the apoptosis-promoting function, and the indirect apoptosis-suppressing function of PTM. Means peptide. Examples of such a protein or peptide include a protein represented by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 in which one or more amino acids have been deleted, substituted or added. included. Here, the number of amino acids to be deleted or the like is not particularly limited, but is usually 20 or less, preferably 10 or less, and particularly preferably 5 or less. The “peptide having a function equivalent to that of PTA1” also includes a peptide fragment obtained from PTA1 and having the above-described necrosis-suppressing function and the like. Examples of such a fragment include peptides represented by the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 (peptides substantially the same as these peptides). Here, “substantially the same peptide” refers to an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 4 or SEQ ID NO: 5, and A peptide that has all or part of the necrosis-suppressing function, apoptosis-promoting function, and indirect apoptosis-suppressing function. Although the number of amino acids to be deleted is not particularly limited, it is usually 5 or less, preferably 3 or less, and particularly preferably 1. In addition, the peptide represented by the amino acid sequence corresponding to SEQ ID NO: 4 in FIG. 2 (the amino acid sequence located in the same column as the amino acid sequence of the rat active body in FIG. 2) is the “substantially identical” Peptides. "

PTA1 indirectly suppresses apoptosis in the presence of microglia, but does not suppress apoptosis in the absence of microglia (conversely, promotes apoptosis). Therefore, depending on the mode of use, the nerve cell death inhibitor of the present invention inhibits only necrosis and inhibits both necrosis and apoptosis. May be controlled.

The nerve cells to which the nerve cell death inhibitor of the present invention is applied are not particularly limited, and can be used to suppress cell death of neurons derived from the cerebral cortex, striatum, midbrain, hippocampus, cerebellum, spinal cord, visual organ, etc. Applicable.

PTA1 can be used not only as a nerve cell death inhibitor, but also as a therapeutic agent for diseases associated with nerve cell death. As used herein, the term “disease associated with neuronal cell death” refers to a disease associated with apoptosis, necrosis, or both. Such diseases include those caused by ischemia, such as stroke, traumatic brain injury, glaucoma, diabetic retinopathy, or compression injury during retinal detachment treatment. Includes diseases caused by the cause or diseases whose cause is not well understood, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis. The term “treatment” used herein includes not only the case where the disease is completely cured, but also the case where the disease condition is reduced or the disease condition is prevented from worsening.

The therapeutic agent for a disease associated with nerve cell death of the present invention is formulated by mixing PTA1 with a pharmaceutically acceptable carrier or diluent according to a known method. Formulations containing suitable carriers and diluents and proteins are described, for example, in Remington's Pharmaceutical Sciences.

With respect to PTA1, the dosage form suitable for administration is not particularly limited, but is preferably prepared as an injection as in the case of a pharmaceutical composition containing many proteins already used as a medicament. More specifically, an injection is prepared by dissolving PTA1 in an appropriate solvent such as water, physiological saline, or an isotonic buffer. At that time, it can be prepared by adding polyethylene dalicol, glucose, various amino acids, collagen, albumin, etc. as a protective material. It is also possible to embed the polypeptide in an inclusion body such as ribosome and administer it.

When PTA1 is used for the treatment of the above-mentioned diseases, the dose varies depending on the age, weight, disease state, administration route, and other factors of the subject, but can be easily determined by the administering physician as appropriate. For example, when administered intravitreally for the treatment of glaucoma, the dose is about 0.0012 mg to 0.012 mg once; when administered intraventricularly for the treatment of stroke, the daily dose is about 0 012 mg to 1.2 mg. The administration method of PTA1 is not particularly limited, and various administration methods that are actually used at present can be adopted for intracerebral administration. An example of such an administration method is intracisternal administration. Intracisternal administration is advantageous in that it does not damage the brain parenchyma. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram schematically showing the effects of PTA1 on nerve cells and microglia.

Figure 2 shows the amino acid sequence of PTA1 actually obtained from organisms such as humans, rats, and mice. (The right end of the amino acid sequence in Figure 2a and the left end of the amino acid sequence in Figure 2b are linked. The right end of the amino acid sequence in Figure 2b is connected to the left end of the amino acid sequence in Figure 2c.)

FIG. 3 is a view showing the amino acid sequence of PTA1 derived from human, rat, and mouse.

FIG. 4 is a diagram showing a step of purifying a protein having a survival activity on cultured neurons.

FIG. 5 is a diagram showing the results of reversed-phase HPLC analysis of a digest of a protein having survival activity and Lysyl endopeptidase.

FIG. 6 is a graph showing the relationship between the survival activity of nerve cells and the amount of rat rPTM coated.

FIG. 7 is a graph showing the relationship between the survival activity of neurons derived from cerebral cortex, striatum, midbrain, or hippocampus and the amount of rat rPTAl coated.

FIG. 8 is a graph showing the relationship between the survival activity of nerve cells and the site of deficiency of rat rPTAl added during nerve cell culture.

FIG. 9 is a fluorescence micrograph of nerve cells cultured in the presence or absence of rat rPTAl.

FIG. 10 is a transmission electron micrograph of nerve cells cultured in the presence or absence of rat rPTAl.

Fig. 11 shows intracellular ATP of neurons cultured in the presence or absence of rat rPTAl. It is a figure which shows a time-dependent change of content.

FIG. 12 is a graph showing the effect of the addition of Cal C or U-73122 on the change in intracellular ATP content of neurons cultured in the presence or absence of rat rPTAl.

FIG. 13 is a graph showing the effect of the addition of Cal C or U-73122 on the 2-DG uptake effect of neurons cultured in the presence or absence of rat rPTAl.

FIG. 14 is a fluorescence micrograph showing the position of GLUT1 or GLUT4 in neurons cultured in the presence or absence of rat rPTAl.

FIG. 15 is a fluorescence micrograph showing the position of cytochrome c in nerve cells cultured in the presence or absence of rat rPTAl.

FIG. 16 is a diagram showing regulation of Be1-2 family protein expression by rat rPTAl as an apoptosis induction mechanism.

FIG. 17 is a diagram showing the effect of adding a neurotrophic factor on the survival activity of nerve cells by rat rPTM.

FIG. 18 is a fluorescence micrograph showing changes in the position of cytochrome c in neurons caused by rat rPTAl and changes due to coexistence with BDNF.

FIG. 19 is a diagram showing the time-dependent change in the amount of PTA1 in cells and culture supernatant. FIG. 20 is a graph showing the time course of the expression level of IL-6 mRNA in microdial and nerve cells cultured in the presence or absence of rat rPTAl.

FIG. 21 shows a light microscope and a transmission electron micrograph of a retinal cell layer prepared from a mouse subjected to an ischemic treatment after the vitreous administration of the mouse PTA1 AS-0DN. FIG. 22 is an optical microscope and a transmission electron micrograph of a retinal cell layer prepared from a mouse to which vitreous administration of mouse rPTAl was performed before or after ischemia.

FIG. 23 is a photomicrograph of a forehead fragment prepared from a mouse subjected to carotid ischemia treatment after intraventricular administration of mouse rPTAl.

FIG. 24 shows the effect of intracerebroventricular administration of mouse rPTAl on latency during step-through in mice treated with carotid ischemia. BEST MODE FOR CARRYING OUT THE INVENTION Itoda

Hereinafter, the present invention will be described in more detail with reference to Examples.

First, experimental materials and methods used in this example will be described.

Primary culture

Primary culture of cerebral cortex of embryonic day 17 embryo rat was performed according to the method described in Sasaki Y. Fukushima N. Yoshida A. and Ueda H. Cellular and Molecular Neurobiology (1998) 18, 487-496. The dispersed cells were spread on a 96-well culture dish previously coated with poly-DL-orditin (manufactured by Shimadama), an 8-well Lab-Tek ^™ chamber, 3.5 and 9 cm culture dishes, and incubated at 37 ° C. And cultured in 5% carbon dioxide. The culture supernatant was cultured at a density of ⁵ × 10 ⁵ cels / cm ² (high density) for 3 days, and the supernatant was filtered through a Mixx Fil Yuichi (Millipore) and stored at -20 until use. Was. The supernatant is coated by adding it to the low-density (1 × 10 ⁵ cels / cm ² ) culture, or by adding it to the culture plate immediately before the culture, incubating at 25 ° C for 2 hours, and then washing twice with PBS. It depends.

Lactate dehydrogenase release experiment

Cytotoxicity was determined by measuring the activity of cytosolic lactate dehydrogenase (LDH) released from the injured cells into the culture supernatant using a cytotoxicity detection kit from Roche Molecular Biochemicals (Mannheim, Germany). It was determined by measuring according to the protocol described in Specifically, it is as follows.

Seed 3.2 x 10 ⁴ cel ls / 100 _d / well of 17-day embryonic cerebral cortical cells on a 96-well culture plate, and after a certain period of time from the start of culture, remove 100 1 culture supernatant and add 100 l Triton X-100 was added to make the total amount 200 l. Transfer 100 n1 to 96-well plate, add 1001 of kit reaction solution, incubate in the dark at room temperature for 90 minutes, measure absorbance at 450 wavelength, and determine maximum LDH release. . On the other hand, to determine the amount of LDH released in the culture supernatant, transfer the culture supernatant (50 1) to a 96-well plate, add 50 1 of 2¾ Triton X-100, add the kit reaction solution 100 1 and then absorb. It was determined by photometry and evaluated as a percent relative LDH release relative to the maximum LDH release. .

WST-8

The viability of the vesicles 2- (2-me thoxy-4-η it ropheny 1) -3- (4-nitropenyl) -5- (2, 4-disulfophenyl) -2H-t etrazolium, mono sodium salt (WST-8) Using a kit (Dojindo Laboratories, Tokyo), experiments were performed according to the attached protocol. WST-8 was incubated at 37 ° C for 3 hours before colorimetry. The surviving activity was determined as a percentage (percent) of the activity immediately after the start of the culture.

'' One-dye exclusion test

Nerve cell death was assessed by reduced trypan blue exclusion from cells. To the cultured cells, 0.4% trypan solution of the same volume as the medium was added, left for 5 minutes, washed twice with ice-cold PBS, and fixed with 4% paraformaldehyde dissolved in PBS at room temperature for 30 minutes. The percentage of cell death was evaluated as a percentage (%) of total cells. Measurement of intracellular ATP level

The luciferin luciferase method was measured using an ATP quantification kit (Molecular Probes, Eugene, OR). Using 2 × 10 ⁶ whole cells, the cells were washed twice with ice-cold PBS and suspended in a cell diluent solution (100 mM Tris-HC1 pH7.75, 4 mM EDTA). 0.5 mM luciferin, 1.25 g / ml luciferase and 1 mM DTT were mixed in a 201 cell suspension. Luciferin-luciferase bioluminescence measurements were performed using LUMAT LB 9507 from EG & Gberthold (BadWildbad, Germany). Annexin V binding and PI staining experiments

Apoptosis and necrosis were evaluated using the AnnexinV-FLU0S staining kit from Roche Molecular Biochemicals and Provided 'iodide (PI) from Sigma, respectively. Cerebral cortical cells were cultured in an 8-well Lab-Tek ^™ chamber, and after a certain period of time, annexin V fluorescein and 10 Mg / ml PI were added, and incubated at room temperature for 15 minutes. Thereafter, the cells were washed with ice-cold PBS and fixed with 4% PFA for 30 minutes at room temperature. Fluorescence was measured using a Carl Zeiss LSM410 fluorescence microscope.

TUNEL analysis

DNA fragmentation in cells showing apoptosis was measured histochemically using the terminal deoxyribonucleotidyl transf erase-mediated dUTP-biotin nick end labeling (TUNEL) method. 8 Ueru of Lab- culture in Tek ^TM chamber one Incubate the cultured cells with 10 g / ml PI at 37 ° for 30 minutes, wash twice with ice-cold PBS and fix with 4% PFA, and then permeabilize with 50% followed by 100% methanol for 5 minutes. Then, treated with Roche Molecular Biochemicals' TUNEL solution at 37 ° (treated for 1 hour, washed twice with PBS, block solution at room temperature for 1 hour (1% ゥ serum albumin dissolved in PBS). , H7.4) and incubated with streptavidin-fluorescein isothiocyanate (FITC) (1: 100; Vector Laboratories, Burlingame, Calif.) For 1.5 hours at room temperature, followed by fluorescence microscopy. Immunocytochemistry of activated caspase 3

Cells cultured in an 8-well Lab-Tek ^™ chamber were fixed with PFA and permeabilized with 0.1% Triton X-100. Incubate with a blocking solution (2% low-fat milk powder, 2% mi serum albumin, 0.1 ¾ Tween-20 in pH 7.4 PBS) at room temperature for 1 hour, and activate caspase 3 antibody (1: 50; Cell Signaling) and incubation at room temperature for 2 hours. After washing, the cells were incubated with FITC-conjugated anti-Egret IgG antibody (1: 200; Cappel, Aurora) for 4 hours at room temperature, and observed with a fluorescence microscope. Cultured cerebral cortex cells were fixed by incubating with 0.1% phosphate buffer (pH 7.4) and 0.5% dartal aldehyde for 1 hour at room temperature, and then incubated with 1% osmium tetroxide for 1 hour at room temperature. Post fixation was performed. Subsequently, after dehydration with alcohol, Epon812 was embedded, and ultra-thin sections of 80 nm were prepared using Ultracut S (Leica, Austria). The sections were stained with uranium acetate and lead citrate for 30 and 5 minutes, respectively, and observed with an electron microscope (JEM-1210; JEOL, Tokyo, Japan).

Immunoplot analysis

Samples subjected to SDS-polyacrylamide electrophoresis using a 12% polyacrylamide gel were anti-caspase 8 and 9 antibodies (1: 500; StressGen, Victoria, Canada) and anti-active caspase 3 antibody (1: 500; Cell) as primary antibodies. Signaling, Tokyo, Japan), anti-Bax, anti-Bim, anti-Bd-2, anti-Bd-xL antibodies (1: 1000; Santa Cruz) were used for immunoblot analysis. For detection of the immunoreactive band, horseradish peroxides were measured using an enhanced chemiluminescent substrate (Super Signaling Substrate) of Pierce Chemical (Rockford, IL). 2 - Dokishi - D- ^[3 H] glucose uptake experiments

The experiment was carried out as follows in accordance with a modified version of Koivisto's method.

Cerebral cortical cells were cultured in 6-well plates containing DMEM / F-12 medium containing 17.5 mM glucose. The plates used were those coated with recombinant small PTA1 and those not coated. After a certain time from the start of culture

[ ³ H] -2-DG (1 Ci / well, 10 nM) was added, and the mixture was further incubated at 37 ° C. for 2 hours. Then, the supernatant was removed and the cells were washed twice with ice-cold PBS. The cells were lysed with a 0.5 M NaOH solution of IOOI, neutralized with 0.5 M hydrochloric acid, and the radioactivity was measured with a liquid scintillation counter.

PKC kinase activity measurement

Cultured cells are suspended in a buffer containing 50 mM Tris-HCl, pH 7.5, 0.3% j3-mercaptoethanol, 5 mM EDTA, 10 mM EGTA, 50 g / ml PMSF, 10 mM benzamidine, and then glycerol of the same volume And homogenized on ice. The PKC activity was measured using the protein kinase C enzyme activity measurement system of Amersham Pharmacia Biotech (Piscataway, NJ) and according to the attached protocol. The culture sample [S] is mixed with a substrate (0.3 mg / ml La-phosphatidyl-L-serin) that is phosphorylated by PKC and mixed with 50 mM Tris-HCl (pH 7.5), 30 mM dithiothreitol, and [r- ³² [P] ATP and magnesium-incubated with ATP buffer for 37 minutes for 15 minutes. Maximum Maximum PKC activation [Max] was measured by adding 24 rag / ml phorbol 12-myristate 13-acetate and 12 mM calcium acetate to the above composition. Background value [B] was measured without the substrate. The reaction was stopped by adding a stop solution, and the solution was adsorbed on peptide-bonded paper. The bound paper was washed twice with 5% acetic acid and the incorporated ³² P radioactivity was measured by liquid scintillation counting. PC activity was determined by the following formula. PKC activation (%) = ([S]-[B]) / ([Max]-[B]) 100

Statistical analysis

After performing multidimensional analysis of variance (AN0VA) analysis, statistical analysis was performed using Student's t-test. Significant differences were based on a 0.05% risk level, and all results were expressed as mean + standard error.

[Example 1] Protein structure determination and preparation of recombinant protein Purification of a viable protein from the cerebral cortical neuron culture supernatant of the 17-day embryo of the rat by anion exchange chromatography, gel filtration, and SDS-PAGE, yielding a molecular weight of approximately 20 kDa And two proteins of about 22 kDa (NDI20 and NDI22) were obtained (FIGS. 4a and b). The survival activity was evaluated using WST-8 Atsushi.

When the resulting two types of proteins were digested with lysyl endopeptidase, a partial peptide having the KEWEEAE sequence was obtained (Fig. 5). Also, NDI20 could be detected stably even when the extraction conditions were changed, but NDI22 could not be detected in some cases. From this, NDI22 was expected to have another small molecule adsorbed to NDI20, and NDI20 was regarded as the active form. Since NDI 20 could be estimated to be PTA1 from the database, rat and mouse cDNA clones were isolated by RT-PCR, expressed in Escherichia coli, and extracted with acidic phenol and biophoresis (Atoichi Co., Ltd .; Recombinant PTAU or less (referred to as “rPTAl”) was prepared using AEP-820). The rPTAl prepared in this manner exhibited the same electrophoretic mobility as the original NDI20, and the molecular weight in the mass spectrum (VOYAGER; (MALDI) PerSeptive Biosystems) was also consistent.

[Example 2] Protective effect of rat rPTAl on neuronal cell death

The cerebral cortical neurons of the 17-day embryo of the rat were cultured under serum-free conditions in the presence or absence of rat rPTAl, and the survival activity and cytotoxicity 12 hours after the start of the culture were evaluated. The culture was performed under two conditions: low-density culture (lxlO ⁵ cells / cm ² ) and high-density culture (5xl0 ⁵ cells / cm ² ) (hereinafter, low-density and high-density unless otherwise specified). The culture shall be performed at the above-mentioned density.) The culture in the presence of rat rPTAl was performed by coating rat rPTAl at a dose of 3 to 50 Pino1 / cm ^{2 on} a 96-well culture dish in advance, and sprinkling nerve cells thereon. Survival activity was evaluated by WST 8 assay, and cytotoxicity was evaluated by LDH release. In addition, the time course of the survival activity and the degree of cytotoxicity when cultured in the presence of 25 pmol m ² of rPTAl was examined. The above results are shown in Figs.

As shown in Fig. 6A, when the cells were cultured in the absence of rat rPTAl (V in the figure), the viability decreased to about 40% of that at the start of the culture. The culture improved the survival activity in a dose-dependent manner. Rat rPTAl also reduced the degree of cytotoxicity in a dose-dependent manner (Fig. 6C).

Rat 17 day embryo cortex, striatum, midbrain, and neural cells prepared from hippocampus, serum-free conditions, and low-density cultured in the presence of rat rPTAl 0~50pmol / cm ^2, after 12 hours Survival activity (by WST-8 Atsushi) was evaluated. In addition, dorsal root ganglion (DRG) cells and cerebral cortical neurons were cultured at low density in the presence of rat rPTAl under serum-free conditions, and the cell death rate after 12 hours was examined using a trypable monochromic exclusion test. . Figure 7 shows the above results.

As shown in FIG. 7A, the improvement of the survival activity by rat rPTAl was observed not only in the cerebral cortex but also in neurons derived from the striatum, midbrain, and hippocampus. Neurodegeneration in the cerebral cortex and hippocampus is a brain region associated with Alzheimer's disease and senile dementia. It is known that neurodegeneration in the striatum is associated with Huntington's disease and that in midbrain degeneration is associated with Parkinson's disease. Therefore, rat rPTAl may be effective in such a wide range of neurodegenerative diseases.

As shown in FIG. 7B, in cerebral cortical neurons, cell death rate was reduced in a dose-dependent manner by culturing in the presence of rat rPTAl, but no decrease was observed in DRG cells.

[Example 3] Protective effect of rPTAl deletion mutant on neuronal cell death

Full length of rat rPTAl already cloned using primers corresponding to each region of the deletion mutant to produce a protein expression clone fused with dalhithion S-transferase (GST) and rat rPTAl deletion mutant PCR was performed using the clone of type III. The obtained PCR product was cloned into PGEX-5X-1 vector. The deletion mutant GST fusion protein was expressed by a conventional method, and purified from the bacterial cell extract using a Darwin's Thion-Sepharose column.

The rPTAl deletion mutant thus prepared was fractionated by electrophoresis on 12% SDS-PAGE, and visualized by Coomassie brilliant blue (CBB) staining.A band of the expected size was observed. Therefore, the survival activity of cerebral cortical neurons of rat 17-day embryos was evaluated using these (Fig. 8A). Survival activity by adding 25 pmol / cm ² rPTAl deletion mutant was 12 hours after the start of culture under serum-free low-density culture conditions. It was measured later. The result is shown in FIG. 8B.

As shown in Fig. 8B, the mutants in which the 48th amino acid was deleted from the N-terminus of rPTAl and the mutants in which the 79th to 112th amino acids were deleted were comparable to the full length of rPTAl. Improved survival activity, but mutants deleted at amino acids 68 and 86 from the N-terminus and mutants deleted at amino acids 30 to 112 and amino acids 58 to 112 The increase in body viability was markedly diminished.

From these facts, it is expected that the active form of rat rPTAl is the 49th to 78th amino acid region. The amino acid sequence of this 49-78 fragment is shown in SEQ ID NO: 4. In human PTA1, the amino acid sequence of the fragment corresponding to this fragment is shown in SEQ ID NO: 5.

[Example 4] Neuronal death mode switch by rat rPTAl

8 Lab-Tek ^TM chamber one Ueru seeded rat 17 day embryo cortical neurons, under serum-free conditions, rats rPTAl (25 pmol / cm ²⁾ and low-density and high-density cultures the presence or absence, 3 After 24 hours, the state of the cells was observed with a fluorescence microscope. Cells were stained by three methods: (1) PI staining and Annexin V staining, (2) PI staining and activated caspase 3 immunofluorescent staining, and (3) PI staining and TUNEL staining. The results are shown in FIGS. 9a to c, e to g, and i to k. The number of cells stained by each staining method is shown in FIGS. 9d, h, and i.

As shown in these figures, when low-density culture was performed in the absence of rat rPTAl, red fluorescence was observed by PI staining, which is an indicator of necrosis, but green fluorescence by Annexin V, which is an indicator of apoptosis. Was not observed. Conversely, in the high-density culture and the low-density culture in the presence of rat rPTAl, red fluorescence as an indicator of necrosis was not observed, and green fluorescence as an indicator of apoptosis was observed. Similar results were confirmed by PI and activated caspase-3 immunofluorescent staining, and by PI and TONEL staining. From these facts, it became clear that rat rPTAl has the effect of converting neuronal necrosis to apoptosis.

[Example 5] Neuronal cell death mode switch action of rat rPTAl by transmission electron microscope observation

Rat Π day embryonic cerebral cortical neurons were cultured under serum-free conditions in rat rPTAl (25 pmol / cm ² ). The cells were cultured at low or high density in the presence or absence, and the state of the cells 12 hours after the start of the culture was observed with a transmission electron microscope. The results are shown in FIG.

As shown in FIG. 10, in cells cultured at low density in the absence of rat rPTAl, damage to the cell membrane, a remarkable decrease in electron density in the cytoplasm, swelling of mitochondria, and the like were observed. On the other hand, when low-density culture was performed in the presence of rat rPTAl, cell membrane damage ゃ mitochondrial swelling was not observed, but phenomena observed in apoptosis such as nuclear fragmentation and nuclear chromatin condensation were observed. Was done. Such a phenomenon was observed even in high-density culture.

[Example 6] Inhibitory effect of rat rPTAl on reduction of ATP content as a mechanism of necrosis

The cerebral cortical neurons of the rat per day embryo were cultured under the same conditions as in Example 5, and the time-dependent changes in the ATP content in the cells were examined. The results are shown in FIG.

As shown in FIG. 11, when low-density culture was performed in the absence of rat rPTAl, the intracellular ATP content rapidly decreased to almost zero level after 18 hours. However, in high-density culture and low-density culture in the presence of rat rPTAl, the decrease in intracellular ATP content was significantly suppressed.

[Example 7] Mechanism of phospholipase C and protein kinase C of necrotic inhibitory effect of rat rPTAl

Rat 17-day embryonic cerebral cortical neurons were cultured at low and high density under serum-free conditions, and the intracellular ATP content 6 hours after the start of the culture was examined. The culture was performed in the presence or absence of rat rPTAl (25 pmol / cm ² ), and calphosphin C (Cal 0 or U-73122 (U22) was added to 1 / i M immediately after the start of the culture. The results are shown in Fig. 12 A. In addition, calhostin C is a protein kinase C (PKC) inhibitor, and ϋ-73122 is a phospholipase C (PLC) inhibitor. As shown in Fig. 12A, rat rPTAl By culturing in the presence, the decrease in ATP content was suppressed, but this effect was abolished by the addition of Cal C and U-73122.

Furthermore, the mechanism of suppression of the decrease in intracellular ATP content by rat rPTAl was investigated. As shown in Figure 12B, culturing in the presence of rat rPTAl suppressed the decrease in ATP content, but this effect was due to the inhibitor of glucose uptake, 2-dexoxy. It disappeared by the addition of ciglucose (2-DG), iodoacetic acid (IA), a glycolytic inhibitor, and oligomycin (01 igo), an inhibitor of ATP production in mitochondria. Therefore, suppression of the decrease in intracellular ATP content by rat rPTAl suggests an increase in the ATP production system that occurs after uptake of dalcos by cells.

[Example 8] Rat rPTAl suppresses glucose uptake reduction

Rat Π The embryonic cerebral cortical neurons were transformed with rat rPTAl in the presence or absence of serum.

(25 pmol / cm ² ) in the presence or absence of low-density and high-density cultivation, and 2 hours after the start of cultivation, 2-dexoxy-D- [¾] glucose uptake was evaluated. Figure 13 shows the results.

As shown in Fig. 13, the glucose uptake of 2-dexoxy-D- [] glucose was reduced to 10% or less in the culture under serum-free conditions compared to the culture in the presence of serum. By culturing in the presence of rat rPTAl, 2-dexoxy-D- [¾] glucose uptake recovered to about 65% of that in culture in the presence of serum. However, the effect of suppressing the decrease was also eliminated by adding Cal C or II-73122.急速 It was clarified that the rapid decrease in the content was associated with a decrease in the glucose uptake effect, suggesting that the primary reaction in the rat APT content suppression effect of rat rPTAl was due to the suppression of the glucose uptake effect. Was done.

[Example 9] Effect of rat rPTAl on cell membrane localization of glucose transporter

Cerebral cortical neurons of the 17-day embryo of the rat were cultured at low density, and the state of the cells 2 hours after the start of the culture was observed with a fluorescence microscope. The culture was performed in the presence or absence of rat rPTAl (25 mol / cm ² ) in the presence or absence of serum (10% FBS) or serum, and Cal C was added to 1 M immediately after the start of the culture. Cells were also stained with a primary antibody recognizing GLUT1 or GLUT4 (these are glucose transporters) and a fluorescently labeled secondary antibody recognizing the primary antibody. The results are shown in FIG.

As shown in FIG. 14, cell surface localization of GLUT1 and GLUT4 was observed in cells cultured in the presence of serum but not in cells cultured in serum-free conditions. In cells cultured in the presence of rat rPTAl, GLUT1 and GLUT4 were observed to be localized in the cell membrane even in the absence of serum. Disappeared. From the results of Examples 6-9, rat rPTAl is mediated by a receptor expected to be present on the cell membrane, followed by activation of PLC and PKC, localization of GLUT1 and GLUT4 to the cell membrane, promotion of glucose uptake, It has been shown to drive mechanisms such as increased ATP production.

[Example 10] Release of cytochrome C (Cyto C) from mitochondria as a mechanism of induction of apoptosis mechanism by rat rPTAl

The cerebral cortical neurons of the 17-day embryo of the rat were cultured at low density and high density under serum-free conditions, and the state of the cells 12 hours after the start of the culture was observed with a fluorescence microscope. The culture was performed in the presence or absence of rat rPTAl (25 pmol / cm ² ), and Cal C was added to 1 M immediately after the start of the culture. Cells were double-stained with CMRXos (red fluorescence, binding to mitochondria) and anti-Cyto C antibody (green fluorescence, binding to Cyto C). Anti-Cyto C antibody was recognized by a fluorescently labeled secondary antibody. The results are shown in FIG.

As shown in Fig. 15, when low-density culture was performed in the absence of rat rPTAl, green fluorescence was observed at the mitochondrial position, and Cyto C was not released, but was localized in the mitochondria. . On the other hand, in high-density culture and low-density culture in the presence of rat rPTAl, green fluorescence was not observed and Cyto C was released from mitochondria. In addition, such release of Cyto C was suppressed by Cal C.

This example demonstrates that rat rPTAl aggressively induces apoptosis, since Cyto C release is thought to be a mechanism of apoptosis induction because it triggers subsequent caspase activation. .

[Example 11] Regulation of Bcl-2 family protein expression by rat rPTAl as an apo! Cis mechanism induction mechanism

The cerebral cortical neurons of the 17-day embryo of the rat were cultured at low density under serum-free conditions, and the cells were collected over time from the start of the culture, and analyzed by the immunoblotting method. The culture was performed in the presence or absence of rat rPTAl (25 pmol / cm ² ), and Cal C and U73122 were added to M immediately after the start of the culture.

Rat rPTAl treatment at 25 pmol / cm ² in low-density culture causes a time-dependent increase in Bax expression that promotes Cyto C release (Figure 16A, B), and also increases Bim expression with a similar function (Fig. 16B). In contrast, Bd-2 acts to suppress Cyto C release And the expression of Bd-xL was reduced (Fig. 16A, B). The effect of increasing Bax by rat rPTAl was blocked by U73122 and Cal C (Fig. 16C). Based on the results of Examples 10 and 11, the mechanism of rat apoptosis induction by rPTAl promotes Cyto C release from mitochondria through regulation of Bd-2 family protein expression via PLC and PKC, as well as suppression of necrosis. It has been proved that this leads to apoptosis mechanism such as activation of caspase system.

[Example 1.2] Enhancement of cell death inhibitory effect of rat rPTAl by neurotrophic factor Rat cerebral cortical neurons of 17-day embryo were cultured at low density under serum-free conditions, and survival activity 12 hours after the start of culture ( WST-8 Atsushi) was evaluated. The culture was performed in the presence or absence of rat rPTAl (25 mol / cm ² ), and NGF, BDNF, bFGF, or IL6 was added to 100 ng / ml immediately after the start of the culture. Figure 17 shows the results.

As shown in Figure 17, the addition of neurotrophic factor enhanced the cell death inhibitory effect of rat rPTAl. However, these neurotrophic factors alone had no effect on suppressing cell death.

[Example 13] Inhibition of apoptosis-inducing effect of rat rPTAl by BDNF Rat cerebral cortical neurons of 17-day embryo were cultured at low density under serum-free conditions, and the state of the cells 12 hours after the start of culture was determined. Observed with a fluorescence microscope. The culture was performed in the presence or absence of rat rPTAl (25 pmol / cm ² ), and BDNF was added immediately after the start of the culture so as to be 100 ng / ml. The cells were stained with CMRXos (red fluorescence, binding to mitochondria) and double staining with an anti-Cyto C antibody (green fluorescence, binding to Cyto C), which was fluorescently labeled and recognized as a secondary antibody. Was. The results are shown in FIG.

Cyto C releasing action of rat rPTAl was completely suppressed by the addition of BDNF. Examples 12 and 13 demonstrate that rPTAl inhibits necrosis of cerebral cortical neurons and switches to an apoptotic form that is protected by neurotrophic factors.

[Example 14] Release of endogenous PTA1 from nerve cells due to serum-free stress Rat cortical neurons of 17-day embryos were cultured at high density in the absence of serum or serum to determine the amount of endogenous PTA1 released. The intracellular content of PTA1 was quantified. Endogenous PTA1 release was concentrated and purified by extraction of the culture supernatant with acidic phenol, developed by SDS-PAGE, and visualized and quantified by the Brustin method. On the other hand, the intracellular content was directly extracted and quantified similarly. The results are shown in FIG.

As shown in FIG. 19, in the culture under serum-free conditions, endogenous PTA1 release was observed in a time-dependent manner, while the intracellular content decreased in a time-dependent manner. Such endogenous PTA1 release was not observed in the culture in the presence of serum. This suggested that this release was serum-free or starvation stress-induced non-classical release.

[Example 15] Increase in microglial neurotrophic factor gene expression by rat rPTAl

Microglia prepared from the cerebrum of a 17-day-old rat embryo were cultured, and rat rPTAl (25 pmol / cm ² ) was added under the condition that the serum was temporarily made 1%, and the amount of IL-6 mRNA (RT-PCR Over time) was measured. A similar experiment was performed using cerebral cortical neurons from rat 17-day embryos cultured at low density without serum instead of microglia. Also, the amount of IL-6 mRNA was measured one hour after the addition of rat rPTAl while the amount of rat rPTAl was varied. At this time, the amount of cyclophilin mRNA was also measured for comparison. The above results are shown in Figures 20A, B and C.

Addition of rat rPTAl markedly increased the expression level of the microglial IL-6 gene (Fig. 2 OA). Such an increase in gene expression was observed in the dosage range of 0.1 to 25 pmol / ciii ² (FIG. 20B). In addition, such an increase in gene expression was not observed in nerve cells (FIG. 20C).

[Example 16] Exacerbation effect of retinal ischemia stress-induced neuronal cell death by reduction of endogenous PTA1 content by antisense method

Retinas extracted from ischemia-treated ddY mice were embedded in paraffin to prepare sections of the retinal cell layer. The sections were stained with hematoxylin-eosin and then observed using an optical microscope. Further, a section embedded in epoxy resin was prepared, and the outer granular layer was observed with a transmission electron microscope. For the ischemia treatment, a reperfusion model was used in which retinal ischemia was performed by applying a pressure of 130 mmHg to the anterior chamber of the mouse for 45 minutes and then returning to normal pressure. In addition, three rounds of mouse PTA1 gene 120 hours, 72 hours, and 24 hours before ischemic treatment Antisense oligodeoxynucleotide (AS-0DN, ACT GCC GCG TCT GAC ATG GT) and missense oligodeoxynucleotide (MS_ODN, AGT GCA GCT TCG CAC CTG GT) in vitreous g / ll / -dose Was administered. A photograph of the retinal cell layer section 4 days after the ischemia treatment is shown in FIG. 21B, and a photograph of the outer nuclear layer section 24 hours after the ischemia treatment is shown in FIG. 21F.

In addition, the number of cells contained in each of the optic ganglion cell layer, the inner nuclear layer, and the outer nuclear layer was counted from the microscope image. The results are shown in FIGS. 21C, D, and E.

Fig. 21A shows the protein amount of PTA1 when AS-0 丽 and MS-0DN were administered (concentrated and purified by acidic phenol extraction, developed by SDS-PAGE, and visualized by the Brustin method). .

As shown in FIGS. 21B, C, D and E, the thickness of the retinal nerve cell layer was reduced by the ischemic treatment. The thickness was further reduced when AS-0DN was administered, but no such change was observed when MS-0DN was administered.

[Example 17] Inhibition of retinal ischemic nerve injury by mouse rPTAl

Sections of the retinal cell layer and outer nuclear layer were prepared from ddY mice treated with ischemia and observed with a transmission electron microscope. The ischemic treatment and preparation of the sections were performed in the same manner as in Example 16. 30 minutes before or 24 hours after the ischemia treatment, 0.1 or 1. Opmol mouse rPTAl and 100 pmol Cal C or Herb imyc in A (tyrosine kinase inhibitor) were injected into the vitreous body. Pictures of retinal cell layer sections 4 days after ischemia treatment are shown in FIGS. 22A to G, and photographs of outer nuclear layer sections 24 hours after ischemia treatment are shown in FIGS. 22J to N.

In addition, the number of cells contained in each of the optic ganglion cell layer, the inner nuclear layer, and the outer nuclear layer was counted from the microscope image. The results are shown in FIGS. 22 G, H, and I.

Although ischemia treatment reduced the thickness of the retinal nerve cell layer (FIGS. 22A and B), such a decrease in thickness was suppressed by administration of mouse i-PTAl. The administration of mouse rPTAl was effective before and after the ischemic treatment (Fig. 22C, F), and was effective in both the administration of 0.1 lpniol and the administration of 1. Opmol (Fig. 22). H ~ J). The effect of administration of mouse rPTAl was not affected by Herbimyc in A (FIG. 22E), but was abolished by administration of Cal C (FIG. 22D).

In addition, necrosis was observed in the cells of the outer nuclear layer due to the ischemic treatment (Fig. 22 J, K). When mice were pre-administered with rPTAl, necrosis was suppressed, and no apoptosis was observed (Fig. 22L). When mouse rPTAl was administered together with Cal C, necrosis was observed, and the effect of rPTAl to suppress necrosis was abolished. When mouse rPTAl was administered together with Herb imyc in A, necrosis was not observed and apoptosis was observed.

[Example 18] Inhibition of cerebral ischemic nerve injury by mouse rPTAl

After ligating the carotid artery of the ddY mouse for 30 minutes and reperfusion, 4 days later, a forehead section (1 mm) containing the cerebral striatum or hippocampus area was prepared and stained with TTC. Observed in a mirror. Mice received intraventricular administration of 1 pmol of mouse rPTAl 30 minutes before or 24 hours after ischemia treatment. The results are shown in FIG.

As shown in FIG. 23, when carotid artery ligation was performed without administration of mouse rPTAl, white deficient portions were observed in the striatum and the hippocampus region. However, when mouse rPTAl was administered, such a portion without the pigment was not observed before and after the ischemic treatment, and the injury due to the ischemic treatment was suppressed.

[Example 19] Inhibition of cerebral ischemia-induced learning disorder by mouse rPTAl

After ligating the carotid artery of the ddY mouse for 30 minutes, it was reperfused, and four days later, a step-through passive avoidance experiment was performed. Mice were intracerebroventricularly administered 10 pmol of mouse rPTAl 30 minutes before or 24 hours after the ischemic treatment as in Example 18. Figure 24 shows the results of the Step Through type passive avoidance experiment.

As shown in Figure 24, the ischemia-treated mice showed a significant delay in latency to darkroom movement compared to the sham-treated controls, but when mice were administered rPTAl, Regardless, this latency delay has disappeared. This description includes part or all of the contents as disclosed in the description and Z or drawings of Japanese Patent Application No. 2003-015735, which is a priority document of the present application. In addition, all publications, patents, and patent applications cited in the present invention are incorporated herein by reference in their entirety. Industrial applicability According to the present invention, apoptosis and necrosis of nerve cells can be suppressed. This makes it possible to treat diseases involving neuronal death, such as stroke and glaucoma.

Claims

The scope of the claims

1. A nerve cell death inhibitor comprising prothymosin 1 or a protein or peptide having a function equivalent thereto as an active ingredient.

2. The nerve cell death inhibitor according to claim 1, wherein prothymosin 1 is a protein represented by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

3. The nerve cell death inhibitor according to claim 1, wherein the peptide having a function equivalent to that of prothymosin α1 is a peptide represented by the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

4. The nerve cell death inhibitor according to any one of claims 1 to 3, wherein the nerve cell death is apoptosis and necrosis.

5 · An agent for treating a disease associated with neuronal cell death, comprising prothymosin 1 or a protein or peptide having a function equivalent thereto as an active ingredient.

6. The therapeutic agent for a disease associated with nerve cell death according to claim 5, wherein prothymosin αΐ is a protein represented by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

7. The therapeutic agent for a disease associated with nerve cell death according to claim 5, wherein the peptide having a function equivalent to that of prothymosin a1 is a peptide represented by the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

8. The therapeutic agent for a disease associated with nerve cell death according to any one of claims 5 to 7, wherein the nerve cell death is apoptosis and necrosis.

9. The neuronal cell death according to any one of claims 5 to 8, wherein the disease associated with neuronal cell death is stroke, traumatic brain injury, glaucoma, diabetic retinopathy, or compression injury during retinal detachment treatment. For diseases associated with

10. A peptide represented by the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or a peptide substantially identical to these peptides.