Composition for improving memory and cognitive function or preventing or treating neurodegenerative nerve diseases, comprising serpina1e protein as active ingredient

Abstract

The present invention relates to a composition for improving memory and cognitive function or preventing or treating neurodegenerative diseases, the composition comprising SerpinA1e protein as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for preventing, ameliorating, or treating memory impairment, cognitive dysfunction, or neurodegenerative diseases, the composition comprising SerpinA1e protein or a polynucleotide encoding the protein as an active ingredient. The present invention also relates to a method for treating memory impairment, cognitive dysfunction, or neurodegenerative diseases, the method comprising a step for administering a recombinant expression vector, which contains a SerpinA1e protein or a polynucleotide encoding the protein, into a blood vessel of a nonhuman mammal.

Description

Composition for improving memory and cognitive function or preventing or treating brain nerve diseases, containing Serpina1e protein as an active ingredient

[0001] The present invention relates to a composition for improving memory or cognitive function or for preventing or treating brain nerve diseases, comprising SerpinA1e protein as an active ingredient.

[0002] Today, dementia is one of the major neurological diseases, and is considered one of the four major causes of death, following heart disease, cancer, and stroke. The prevalence and incidence rate mainly increase with age. As we enter an aging society due to the increase in average life expectancy caused by the advancement of medical technology, the prevalence and incidence of dementia are rapidly increasing.

[0003] Dementia significantly reduces the quality of life of not only the patient but also their caregivers and those around them due to memory loss and cognitive decline. Therefore, there is a shift in awareness that dementia is no longer simply a disease for individual patients, but that national efforts must be made to prevent, detect early, and treat the disease early.

[0004] Not only patients with degenerative brain diseases like dementia, but also healthy individuals experience neurodegeneration due to age, resulting in memory loss and cognitive decline. Given the rapidly increasing elderly population in Korea, the proportion of this population is expected to increase significantly in the future. Therefore, the development of technologies that can alleviate age-related cognitive and memory decline is urgently needed.

[0005] Cognitive function is the ability to recognize and distinguish objects. Maintaining cognitive function means maintaining normal brain function by controlling various factors that can impair memory or concentration.

[0006] Memory is also closely related to the hippocampus, where plastic changes in neural connectivity based on learning experiences are essential for maintaining normal memory. Furthermore, the presence of neurotransmitters in sufficient quantities facilitates smooth signaling between brain cells. In states of cognitive decline, changes in neural connectivity and neurotransmitter activity decrease.

[0007] One of the major causes of dementia and age-related decline in memory and cognitive function is known to be the loss of neuronal plasticity and associated signaling due to neuronal degeneration. In dementia, this phenomenon is known to be exacerbated by increased activity of acetylcholinesterase (AChE), an enzyme that breaks down acetylcholinesterase, a neurotransmitter. Therefore, AChE inhibitors such as tacrine, donepezil, galantamine, and rivastigmine have been developed as dementia treatments. Age-related neurodegeneration is thought to be caused by increased brain cell death and decreased new neuronal cell production due to aging mechanisms, leading to cognitive decline.

[0008] However, no drug has yet been developed that can suppress brain cell damage or directly regenerate brain cells, and no drug has yet been developed that can effectively improve memory and cognitive function.

[0009] Meanwhile, in the present invention, when SerpinA1e protein was injected into the blood of mice, the SerpinA1e protein was detected in the cerebrospinal fluid by passing through the blood-brain barrier, and it was confirmed that it could increase the production of neurons, the number of neurons, and the plasticity of neurons. Furthermore, by confirming the memory-enhancing effect in normal and aged mouse models, it was determined that the SerpinA1e protein of the present invention can be usefully used as a preventive and therapeutic agent for various neurological diseases including memory, cognitive function, dementia, and age-related cognitive decline, thereby completing the present invention.

[0010] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction or brain nerve disease, comprising SerpinA1e protein or a polynucleotide encoding the protein as an active ingredient.

[0011] Another object of the present invention is to provide a method for treating memory impairment, cognitive dysfunction or neurological disease, comprising a step of administering a recombinant expression vector containing a SerpinA1e protein or a polynucleotide encoding the protein into a blood vessel of a mammal other than a human.

[0012] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction or brain nerve disease, comprising SerpinA1e protein or a polynucleotide encoding the protein as an active ingredient.

[0013] In one embodiment of the present invention, the protein may be composed of an amino acid sequence of sequence number 1.

[0014] In one embodiment of the present invention, the polynucleotide may be composed of a base sequence of sequence number 2.

[0015] In one embodiment of the present invention, the SerpinA1e protein may pass through the blood-brain barrier.

[0016] In one embodiment of the present invention, the composition may increase the production of nerve cells; increase the expression of BDNF (brain-derived neurotrophic factor) protein; and increase the length and number of nerve cell branches.

[0017] In one embodiment of the present invention, the brain disease may be selected from the group consisting of mild cognitive impairment, Parkinson's disease, Huntington's disease, Alzheimer's disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

[0018] In one embodiment of the present invention, the SerpinA1e protein may be a recombinant SerpinA1e protein to which a marker amino acid sequence of SEQ ID NO: 7 for detection and purification of the protein is linked.

[0019] In one embodiment of the present invention, the recombinant SerpinA1e protein may be composed of an amino acid sequence of SEQ ID NO: 3.

[0020] In one embodiment of the present invention, the polynucleotide encoding the recombinant SerpinA1e protein may be composed of the base sequence of SEQ ID NO: 4.

[0021] The present invention also provides a method for treating memory impairment, cognitive dysfunction or neurological disease, comprising a step of administering a recombinant expression vector containing a SerpinA1e protein or a polynucleotide encoding the protein into a blood vessel of a mammal other than a human.

[0022] In one embodiment of the present invention, the protein may be composed of an amino acid sequence of sequence number 1.

[0023] In one embodiment of the present invention, the polynucleotide may be composed of a base sequence of sequence number 2.

[0024] In one embodiment of the present invention, the SerpinA1e protein may have an effect of preventing, improving, or treating memory impairment, cognitive dysfunction, or neurological diseases by passing through the blood-brain barrier; increasing the production of nerve cells; increasing the expression of BDNF (brain-derived neurotrophic factor) protein; and increasing the length and number of nerve cell branches.

[0025] In one embodiment of the present invention, the SerpinA1e protein may be a recombinant SerpinA1e protein to which a marker amino acid sequence of SEQ ID NO: 7 for detection and purification of the protein is linked.

[0026] In one embodiment of the present invention, the recombinant SerpinA1e protein may be composed of an amino acid sequence of SEQ ID NO: 3.

[0027] In one embodiment of the present invention, the polynucleotide encoding the recombinant SerpinA1e protein may be composed of the base sequence of SEQ ID NO: 4.

[0028] The present invention has discovered a novel use of SerpinA1e protein having an effect of preventing, improving or treating memory impairment, cognitive dysfunction or brain nerve disease. The SerpinA1e protein according to the present invention can pass through the blood-brain barrier when injected into a blood vessel, and can induce all of the activities of increasing the production of nerve cells, increasing the expression of BDNF (brain-derived neurotrophic factor) and increasing the length and number of nerve cell branches, and can be used as a new pharmaceutical product that can effectively prevent, improve and treat memory impairment, cognitive dysfunction or brain nerve disease.

[0029] Figure 1 shows an analysis of the expression level of Serpina1 protein secreted into the blood from muscles or other organs other than muscles after exercise in a mouse model (A), and a comparative analysis of the expression levels of SerpinA1a and SerpinA1e proteins, which are Serpina1 family proteins (B).

[0030] Figure 2 shows the results of confirming the detection of the recombinant SerpinA1e protein in plasma and cerebrospinal fluid after blood injection of the recombinant SerpinA1e protein of the present invention into mice.

[0031] Figure 3 shows the results of confirming the memory improvement effect according to the increase in SerpinA1e protein in the blood after intraorbital injection of recombinant SerpinA1e protein and recombinant SerpinA1a protein into mice (*p<0.05, unpaired t-test).

[0032] Figure 4 shows the results of confirming an increase in new neuron generation in the dentate gyrus of the hippocampus by blood injection of the recombinant SerpinA1e protein of the present invention (scale 50 μm, **p<0.01, unpaired t-test).

[0033] Figure 5 shows the results of confirming an increase in the amount of BDNF protein in the hippocampus by blood injection of the recombinant SerpinA1e protein of the present invention (**p=0.0028, unpaired t-test).

[0034] Figure 6 shows the results of confirming the generation of new neurons in the dentate gyrus of the hippocampus by blood injection of the recombinant SerpinA1a protein of the present invention (p>0.05, unpaired t-test).

[0035] Figure 7 shows the results of confirming an increase in the length and number of branches of cultured nerve cells following treatment with the recombinant SerpinA1e protein of the present invention (scale 5 μm, *p<0.05 ***p<0.001 ****p<0.0001, unpaired t-test).

[0036] Figure 8 shows the results of confirming the effect of recovering cognitive decline (discrimination index) and increasing new neuron generation (BrdU+DAPI+) in the dentate gyrus of the hippocampus in an aged mouse model by blood injection of recombinant SerpinA1e protein. Aged Ctrl represents the control group of aged mice, Aged EX represents the group of aged mice that underwent aerobic exercise, and Aged Serpina1e represents the group of aged mice that were blood injected with Serpina1e protein.

[0037] The present invention is characterized by providing a pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction, or brain nerve disease, which comprises SerpinA1e protein or a polynucleotide encoding the protein as an active ingredient.

[0038] In the present invention, the SerpinA1e (serine (or cysteine) peptidase inhibitor, clade A, member 1E) protein has been studied in relation to liver disease as a physiological activity of the SerpinA1e protein, but there has been no study at all on its relation to memory impairment, cognitive dysfunction, or brain nerve disease.

[0039] In order to develop a new therapeutic agent that can effectively treat memory impairment, cognitive dysfunction, or neurological diseases, the present inventors identified proteins that are secreted from muscles during exercise and whose concentration in the blood increases. As a result of the analysis, it was confirmed that the protein concentration of SerpinA1e is secreted from muscles during exercise and its concentration in the blood increases significantly.

[0040] Accordingly, in order to study the correlation between the blood concentration of SerpinA1e protein and memory enhancement, the inventors of the present invention prepared a recombinant SerpinA1e protein that can be administered intravascularly to a mouse model, and after administering this into the blood vessels of mice, analyzed the location of the recombinant protein and whether it had a memory enhancement effect.

[0041] As a result, it was confirmed that the recombinant SerpinA1e protein administered intravascularly to mice was present not only in the plasma but also in the cerebrospinal fluid.

[0042] In particular, the detection of recombinant SerpinA1e protein in cerebrospinal fluid means that intravascularly administered recombinant protein can pass through the blood-brain barrier, thereby solving the problem of not being able to pass through the blood-brain barrier, which has been a major obstacle in the development of treatments for brain diseases.

[0043] In addition, the experimental group of mice administered the recombinant SerpinA1e protein showed an increase in hippocampus-dependent long-term memory formation ability compared to the control group that was not administered the protein. Interestingly, this memory enhancement effect was not observed in the group administered the recombinant SerpinA1a protein, and was confirmed to be effective only in the group administered the recombinant SerpinA1e protein.

[0044] In addition, in another embodiment of the present invention, it was confirmed that the generation of new nerve cells increased in the dentate gyrus of the hippocampus of mice administered with the recombinant SerpinA1e protein, and the expression of the brain-derived neurotrophic factor (BDNF) protein, known as a protein essential for memory formation, was also found to be significantly increased, and it was confirmed that the length and number of nerve cell branches were also significantly increased.

[0045] The above BDNF (brain-derived neurotrophic factor) is known to play a role in improving memory and cognitive function by promoting the creation of neurons in the hippocampus, the brain tissue responsible for memory. It can also maintain the survival of neurons and promote the growth and differentiation of new neurons and synapses, and plays an important role in areas of the brain particularly damaged by dementia. Recently, dementia treatment technology using BDNF gene therapy is being developed.

[0046] In addition, in the present invention, it was confirmed that when recombinant SerpinA1e protein was administered, the length and number of branches of nerve cells increased, which means that the protein can increase neural circuit plasticity (neural plasticity).

[0047] The above neural plasticity refers to the ability of the brain's nerve cells to grow and change throughout life in response to new stimuli, and refers to the characteristic of the brain changing its shape by building new neural networks in response to new learning or experiences.

[0048] This increase in neuroplasticity has been linked to improved memory and cognitive function, and is also being used to treat and rehabilitate neurological disorders, including dementia and Parkinson's disease.

[0049] In this respect, the inventors of the present invention were able to find that administering the SerpinA1e protein of the present invention can effectively prevent, improve, and treat memory impairment, cognitive dysfunction, or brain nerve diseases.

[0050] Therefore, the present invention is characterized by providing a pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction, or brain nerve disease, which comprises SerpinA1e protein or a polynucleotide encoding the protein as an active ingredient.

[0051] The SerpinA1e protein may be composed of an amino acid sequence of sequence number 1, and in one embodiment of the present invention, a recombinant SerpinA1e protein composed of an amino acid sequence of sequence number 3 with an added amino acid sequence labeled V5 (GKPIPNPLLGLDST) was used.

[0052] Here, the V5 tag amino acid sequence is a V5 epitope tag consisting of 14 amino acids of GKPIPNPLLGLDST, which is used for detection and purification of recombinant proteins and does not affect the activity and properties of the SerpinA1e protein.

[0053] Additionally, the above "polynucleotide" is a polymer of deoxyribonucleotides or ribonucleotides existing in single-stranded or double-stranded form. It encompasses RNA genome sequences, DNA (gDNA and cDNA), and RNA sequences transcribed therefrom, and may include analogs of natural polynucleotides unless otherwise specifically stated.

[0054] The polynucleotide may include not only a nucleotide sequence encoding the amino acid sequence of the SerpinA1e protein, but also a complementary sequence to that sequence. The complementary sequence includes not only a perfectly complementary sequence but also a substantially complementary sequence, and may include a sequence that can hybridize with the nucleotide sequence of the nucleotide sequence encoding the amino acid sequence of the protein, for example, under stringent conditions known in the art.

[0055] In the present invention, the polynucleotide encoding the SerpinA1e protein may be composed of the base sequence of SEQ ID NO: 2, and preferably may be composed of the base sequence of SEQ ID NO: 4 encoding a recombinant SerpinA1e protein to which a marker amino acid sequence called V5 has been added.

[0056] Additionally, the polynucleotide may be included in the composition in the form of a vector containing a polynucleotide encoding a SerpinA1e protein.

[0057] The above vector may be any one selected from the group consisting of, but not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus (AAV) vector.

[0058] The vector containing the above polynucleotide refers to a means for expressing a target gene in a host cell or a target site, and the vector includes elements for expressing the target gene, such as a replication origin, a promoter, an operator, a transcription termination sequence, etc., and may further include an appropriate enzyme site (e.g., a restriction enzyme site) for introduction into the genome of a host cell and / or a selection marker for confirming successful introduction into a host cell and / or a ribosome binding site (RBS), IRES (Internal Ribosome Entry Site) for translation into a protein, etc. The vector may further include a transcription control sequence (e.g., an enhancer, etc.) other than the promoter.

[0059] In the above recombinant vector, the polynucleotide sequence encoding the protein may be operably linked to a promoter. The term "operably linked" refers to a functional linkage between a nucleotide expression regulatory sequence, such as a promoter sequence, and another nucleotide sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleotide sequence.

[0060] In addition, the brain nerve disease that the composition of the present invention can prevent or treat is not limited thereto, but may be selected from the group consisting of mild cognitive impairment, Parkinson's disease, Huntington's disease, Alzheimer's disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

[0061] In the present invention, the term "treatment" is used to mean alleviation or improvement of pathological symptoms, reduction of the area of ​​the disease, delay or alleviation of disease progression, improvement, alleviation or stabilization of the disease state or symptoms, partial or complete recovery, prolongation of survival, and other beneficial treatment results. The term "prevention" is used to mean all mechanisms and / or effects that act on a subject who does not have a specific disease to prevent the development of the specific disease, delay the onset of the disease, or reduce the frequency of the development of the disease.

[0062] The term "pharmaceutical composition" may refer to a molecule or compound that, when administered to a subject, imparts several beneficial effects. Beneficial effects may include enabling diagnostic determination; improving a disease, symptom, disorder, or condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or condition.

[0063] In addition to the active ingredient, the pharmaceutical composition may further comprise one or more auxiliary agents selected from the group consisting of pharmaceutically acceptable carriers, excipients, diluents, fillers, bulking agents, wetting agents, disintegrating agents, emulsifiers (surfactants), lubricants, sweeteners, flavoring agents, suspending agents, preservatives, etc. The auxiliary agents may be appropriately adjusted depending on the dosage form to which the pharmaceutical composition is applied, and one or more auxiliary agents that can be commonly used in the pharmaceutical field may be selected and used. In one specific example, the pharmaceutically acceptable carrier is one that is commonly used in the formulation of drugs, and may be used as a mixture of saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets, and target organ-specific antibodies or other ligands can be combined with the carrier to specifically act on the target organ. Furthermore, the composition can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's literature (Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA).

[0064] The effective amount of the above active ingredient or the pharmaceutical composition can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation. Parenteral administration can mean administration through a route other than oral, such as rectal, intravenous, peritoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, ocular, or subcutaneous, and can be administered by local administration to the affected area, etc. When administered orally, the pharmaceutical composition can be formulated in a form that coats the active ingredient to prevent it from being decomposed in the stomach or protects it from being decomposed in the stomach.

[0065] When the pharmaceutical composition of the present invention is used as a medicine, it may additionally contain one or more active ingredients exhibiting the same or similar function.

[0066] The pharmaceutical composition may be administered in a pharmaceutically effective amount. There are no special restrictions on the dosage, and may vary depending on the absorption rate in the body, body weight, patient's age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The pharmaceutical composition of the present invention is manufactured in consideration of the effective dosage range, and the unit dosage form prepared in this way may be administered multiple times at regular intervals using a specialized dosage method according to the judgment of a specialist who monitors or observes the administration of the drug and the individual's needs, as needed. The dosage of the pharmaceutical composition may be 1 ug / kg / day to 1,000 mg / kg / day, but is not limited thereto. The daily or single dose may be prepared as a single preparation in the form of a unit dose, formulated in an appropriate amount, or prepared by being placed in a multi-dose container.

[0067] The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of treatment for a target disease to be treated.

[0068] In addition, the present invention can provide a method for treating memory impairment, cognitive dysfunction, or brain nerve disease, comprising a step of administering a recombinant expression vector containing a SerpinA1e protein or a polynucleotide encoding the protein into a blood vessel of a mammal other than a human.

[0069] The present invention will now be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0070]

[0071] <Preparation example and experimental method>

[0072] Preparation of experimental animals

[0073] To selectively identify proteins secreted from muscles, Acta1-Cre mice, which express Cre in muscles using a muscle-specific promoter (ACTA1), and LSL-TurboID-ER mice, which can Cre-dependently express biotin ligase (TurboID) in the endoplasmic reticulum, were crossed to obtain ACTA1;TurboID-ER mice. To measure cognitive and brain changes caused by recombinant protein injection, 8-12 week old C57BL / 6 wild type male mice were used. Mice used in the experiment were raised in an experimental animal center that blocks pathogens and maintains a constant environment (12-hour day-night cycle, temperature of 20-24℃, humidity of 30-70%).

[0074]

[0075] Exercise performance and biotin administration to laboratory animals

[0076] Male ACTA1;TurboID-ER mice, 8–12 weeks of age, were randomly divided into control and exercise groups. The exercise group performed voluntary exercise on a wheel five times a week for 12 h during the diurnal cycle for a total of 4 weeks, while the control group was housed in regular cages. To label muscle-derived proteins secreted by exercise, both groups received biotin (24 mg / kg) via intraperitoneal injection 1 h before the diurnal cycle, the start of the exercise period.

[0077]

[0078] Plasma collection

[0079] After anesthetizing the mice in a chamber containing 4% isoflurane, blood was collected from the heart and mixed with ethylene-diamine-tetraacetic acid (EDTA) to a final concentration of 0.5 mM. The blood containing 0.5 mM EDTA was centrifuged at 12,000 rpm at 4°C for 20 min to separate the serum, which was then treated with a protease inhibitor to prevent protein degradation. Avidin magnetic beads (Invitrogen #11205D) were used to separate muscle-derived secretory proteins from the blood of ACTA1;TurboID-ER mice.

[0080]

[0081] Proteomic analysis

[0082] To identify proteins whose secretion increased in the muscles of mice due to exercise, biotinylated proteins (muscle-derived secreted proteins) were identified in the plasma of ACTA1;TurboID-ER mice in the control and exercise groups using an Orbitrap Exlipse Tribrid mass spectrometer (Thremo). Proteins whose quantitative difference increased by more than 1.5 times in the plasma of the exercise group compared to the control group were identified as protein candidates whose secretion increased in the muscles due to exercise. Among the protein candidates, SerpinA1a and SerpinA1e proteins were identified and the level of change in concentration in the plasma of the control and exercise group mice was analyzed.

[0083]

[0084] Intravenous and intraperitoneal administration

[0085] To confirm the cognitive enhancement effect of SerpinA1 protein, a V5-marked amino acid sequence (GKPIPNPLLGLDST) for protein purification and detection was added to SerpinA1a and SerpinA1e, and a recombinant protein containing histidine (6x His) at the end was synthesized (Genscript). 8-12 week-old C57BL / 6 wild-type male mice were anesthetized in a chamber containing 4% isoflurane, and 150 μg of the recombinant protein was injected intravenously (intraorbitally) into the bloodstream at a rate of 1.0 g / day. Injections were performed every 3 days using a 31-gauge insulin syringe, for a total of 9 injections over 24 days. The control group was injected with the same volume of saline solution. In addition, the group injected with both recombinant SerpinA1a and recombinant SerpinA1e proteins received 150 μg of the recombinant protein each, for a total of 300 μg of the recombinant protein injected per injection. To determine the extent of new neuron generation by recombinant SerpinA1a protein (SEQ ID NO: 5) or recombinant SerpinA1e protein (SEQ ID NO: 3), BrdU (5-bromo-2'-deoxyuridine, 50 mg / kg) was administered intraperitoneally for 5 days starting 5 days before the last intravenous injection of the recombinant protein.

[0086]

[0087] CSF (Cerebrospinal fluid) collection

[0088] To determine whether the recombinant SerpinA1e protein is delivered to the brain via the blood vessels, 300 μg of the recombinant protein was injected intravenously (intraorbitally), and cerebrospinal fluid (CSF) was extracted from the cisterna magna 1 hour later. The extracted CSF was centrifuged at 10,000 rpm for 30 seconds to remove cells and debris, and only the CSF in the supernatant was obtained.

[0089]

[0090] Western blot

[0091] Serum and cerebrospinal fluid samples were subjected to SDS-PAGE on 4–20% gels (Bio rad #456-8093) and proteins were transferred to PVDF membranes (Biorad #1704156). Blots were blocked with 3% skim milky (Sigma #70166) and Western blots were performed using the following antibodies: Anti-Serpina1 1:1000 (Thermo #PA5-87677), Anti-V5 1:5000 (Thermo #R960-25), Anti-Rb-HRP 1:10000 (Bio rad #170-6515), and Anti-mouse-HRP (Bio rad #170-6516).

[0092]

[0093] Novel object recognition test

[0094] The novel object orientation test was performed on mice in a cylindrical test chamber (a matte acrylic box, 30 cm in diameter, 28 cm in height) as follows. The test was conducted in three stages: habituation, learning, and testing. During the habituation stage, the mice were exposed to the test environment by freely exploring the test chamber for 5 minutes. In the learning stage, the mice were allowed to stay in the test chamber with two identical objects (A + A) for 5 minutes, and after 24 hours, one of the two objects (A + A) was replaced with a novel object (A + B). The mouse was considered to be exploring the object when its mouth touched or came close to the object. The time taken to explore the familiar and novel objects was recorded separately, and the difference in exploration time for the moved object or novel object was divided by the total exploration time to calculate the 'discrimination ratio'.

[0095]

[0096] Context fear conditioning test

[0097] Eight- to 12-week-old C57BL / 6 wild-type male mice that received nine intravenous (intraorbital) injections began fear conditioning 24 hours after the last injection (the ninth). After one hour of room habituation, they were given a 3-minute exploration period in the fear conditioning chamber. This was done to determine their basal fear level before the stimulus. After the 3-minute exploration period, a 2-second 0.5 mA current was delivered to the floor, followed by an additional 1-minute exploration period. This was done to establish the association between the current stimulus and spatial memory. Twenty-four hours after the fear conditioning, a 3-minute memory test was performed in the same chamber without the current stimulus. The freezing time was used to measure the extent to which the mice remembered the association between the electric stimulus-induced fear and the stimulated space.

[0098]

[0099] Immunohistochemistry

[0100] Mice were anesthetized in a chamber containing 4% isoflurane and perfused with PBS to remove all blood. After perfusion with 4% paraformaldehyde to fix all blood vessels, brain tissue was extracted. The extracted brain tissue was further fixed in 4% paraformaldehyde for one day at 4°C. After removing all moisture from the fixed brain tissue, it was immersed in a 30% sucrose solution and brain blots were prepared using OCT compound. Furthermore, the brain blots were sectioned using a cryostat and stained.

[0101] Brain tissue sections were washed three times with PBS, treated with 2 M HCl, and subjected to antigen retrieval at 37°C for 30 minutes. After washing five times with PBS, blocking was performed using 10% goat serum (Jackson lab #005-000-121), and then stained with Anti-BrdU 1:1000 (Merck #59-14-3) and imaged using a confocal microscope (Nikon #A1 Rsi / Ti-E).

[0102]

[0103] ELISA (Enzyme-linked immunosorbent assay analysis)

[0104] Hippocampal tissue was extracted from the brains of mice that received nine intravenous injections of recombinant SerpinA1e protein. Total cell extracts were obtained from the extracted hippocampal tissue using lysis buffer (1% Triton + 0.1% Tween20 + 100x protease inhibitor cocktail + PBS). In addition, the concentration of BDNF in the hippocampus was measured at a wavelength of 450 nm using a Multiskan Skyhigh (Thermo) instrument using a BDNF ELISA kit (Abnova #KA0331).

[0105]

[0106] Primary neuron culture and processing

[0107] Cells were cultured at 37°C in a 5% CO2 environment. The cerebral cortex was isolated from the embryonic brain on day 17 of pregnancy, washed in HBBSS (Gibco #14170-112), and then treated with 0.25% trypsin while maintaining it at 37°C and reacted for 20 minutes. After removing the trypsin, the cells were washed 10 times with HBSS. Plating media (10% FBS + 1% penicillin / streptomycin + 2 mM glutamine in MEM) was added and the cells were resuspended by pipetting. The cells were filtered through a 40 μm cell strainer and seeded onto plates coated with Poly-L-lysine (sigma #P4707). After seeding, all plate media was removed after 2 hours and the media was replaced with maintenance media (2% B-27 + 1% penicillin / streptomycin + 2 mM glutamine in Neurobasal) to maintain the culture.

[0108] To label neuronal morphology, pCAG-EGFP was transfected on the fourth day of culture. On the sixth day of culture, cells were treated with 10 μg or 100 μg of recombinant SerpinA1e protein, and fixed with 4% paraformaldehyde 24 hours later. The control group was treated with the same amount of saline solution. The fixed cells were imaged with a confocal microscope, and the length and number of neuronal branches were analyzed using the Image J program.

[0109]

[0110] <Example 1>

[0111] Identification of muscle-derived proteins whose expression levels in blood are increased after exercise

[0112] To identify proteins whose secretion from muscles increases due to exercise, a mouse model was used, and after 4 weeks of aerobic exercise, proteins with increased blood concentrations secreted from muscles were analyzed.

[0113]

[0114] As a result, as shown in Fig. 1, it was confirmed that the concentration of Serpina1 protein, a muscle-derived protein in the blood, was significantly increased in the group that exercised compared to the group that did not exercise, and in particular, it was confirmed that the concentration of SerpinA1e, a protein belonging to the Serpina1 family, was very significantly increased.

[0115]

[0116] <Example 2>

[0117] Detection of recombinant SerpinA1e protein in plasma and cerebrospinal fluid after intravenous injection of recombinant SerpinA1e protein.

[0118] The present inventors directly injected recombinant SerpinA1e protein with an added V5-labeled amino acid sequence into the blood vessels of non-exercising normal mice for 4 weeks, and analyzed whether the recombinant protein was detected in the plasma and cerebrospinal fluid. In this case, the recombinant protein was injected intravascularly into the blood vessels of a non-exercising adult mouse model (8-week-old, male) nine times at 3-day intervals for 28 days (see Fig. 3).

[0119]

[0120] As shown in Fig. 2, the recombinant SerpinA1e protein (SerpinA1e-V5 protein) injected into the blood was detected in plasma and also in cerebrospinal fluid. These results indicate that the recombinant protein of the present invention can cross the blood-brain barrier (BBB).

[0121]

[0122] <Example 3>

[0123] Confirmation of memory enhancement effect according to increased plasma concentration of recombinant SerpinA1e protein

[0124] To determine whether increasing the plasma concentration of the recombinant SerpinA1e protein according to the present invention could enhance hippocampal-dependent long-term memory, the recombinant SerpinA1e protein was injected intravascularly into mice, and then a fear conditioning test was performed. A group injected with a saline solution instead of the recombinant protein served as a control group, and a group injected with recombinant SerpinA1a protein instead of the recombinant SerpinA1e protein served as a comparison group.

[0125]

[0126] As a result, as shown in Fig. 3, the group receiving intravenous injection of the recombinant SerpinA1e protein of the present invention showed a significant improvement in memory compared to the control group. On the other hand, the group receiving the same dose and number of injections of SerpinA1a protein, another muscle-derived protein whose concentration in the blood increases due to exercise, did not show any memory-enhancing effect.

[0127] Through these results, the inventors of the present invention were able to determine that the memory improvement effect following administration of the recombinant SerpinA1e protein of the present invention is not an effect that can be exhibited by all proteins belonging to the SerpinA1 family, but is a specific effect of the recombinant SerpinA1e protein of the present invention.

[0128]

[0129] In addition, in the group that received simultaneous injections of recombinant SerpinA1a protein and recombinant SerpinA1e protein, memory formation was significantly improved. This result confirmed once again that the memory improvement effect was due to an increase in SerpinA1e protein in the blood.

[0130]

[0131] <Example 4>

[0132] Increased neurogenesis confirmed by administration of recombinant SerpinA1e protein

[0133] In order to confirm the neurobiological mechanism of the memory enhancement effect following administration of the recombinant SerpinA1e protein of the present invention, the recombinant SerpinA1e protein was administered to a mouse model that did not exercise under the same conditions as in <Example 3>, and then changes in the neural system in the hippocampus were observed.

[0134] As a result, as shown in Fig. 4, it was confirmed that the generation of new neurons was significantly increased in the hippocampal dentate gyrus of mice administered the recombinant SerpinA1e protein of the present invention. In addition, as a result of analyzing the amount of BDNF (brain-derived neurotrophic factor) protein, known as a factor that promotes hippocampal neurogenesis in the brain responsible for memory and learning, it was found that the amount of BDNF protein was significantly increased in the group administered the recombinant SerpinA1e protein of the present invention compared to the control group (see Fig. 5).

[0135] Meanwhile, in the group administered with the recombinant SerpinA1a protein belonging to the SerpinA1 family, it was found that almost no new nerve cells were created, as evidenced by no memory-enhancing effect (see Figure 6).

[0136]

[0137] <Example 5>

[0138] Confirmation of increased branch length and number of neurons following administration of recombinant SerpinA1e protein

[0139] The present inventors confirmed whether the length and number of branches of nerve cells could be increased by administration of the recombinant SerpinA1e protein of the present invention.

[0140]

[0141] As a result, as shown in Fig. 7, the group administered with the recombinant SerpinA1e protein of the present invention showed an increase in the number and complexity of neurites of cultured nerve cells compared to the control group, and it was confirmed that the length and number of nerve cell branches increased in a concentration-dependent manner depending on the treatment concentration of the recombinant protein.

[0142]

[0143] <Example 6>

[0144] Confirmation of the recovery effect of age-related cognitive decline following administration of recombinant SerpinA1e protein

[0145] Furthermore, to determine whether administration of recombinant SerpinA1e protein can restore age-related cognitive decline, the inventors injected recombinant SerpinA1e protein into aged mice and analyzed the level of cognitive function recovery. Specifically, a control group of mice (18-week-old males) with normal aging, a group of aged mice that underwent aerobic exercise (performing aerobic exercise for 4 weeks), and a group of aged mice that received direct intravascular injection of recombinant SerpinA1e protein for 4 weeks were tested on a novel object recognition test, which is an experiment to recognize and remember new objects, and the level of new neurons in the hippocampal dentate gyrus was also analyzed.

[0146]

[0147] As a result, as shown in Fig. 8, the control group of aged mice showed a decline in cognitive ability due to the progression of aging, whereas the group of aged mice that performed aerobic exercise for 4 weeks showed a recovery in cognitive function compared to the control group, and the group administered the SerpinA1e protein of the present invention was able to confirm a better cognitive function recovery ability than the group that performed aerobic exercise.

[0148] In addition, as a result of analyzing the level of new neurons in the hippocampal dentate gyrus, it was observed that the control group of aged mice had a decrease in new neurons in the hippocampal dentate gyrus due to aging, whereas the group of aged mice that performed aerobic exercise for 4 weeks and the group that was administered the SerpinA1e protein of the present invention both showed a significantly greater increase in the generation of new neurons compared to the control group.

[0149] These results indicate that the SerpinA1e protein of the present invention can effectively improve age-related cognitive decline and improve the decline in neuronal cell production due to aging.

[0150]

[0151] Through the above results, the inventors of the present invention confirmed that the recombinant SerpinA1e protein of the present invention can increase the production of neurons, increase the number of hippocampal neurons, increase the plasticity of neural circuits, and improve cognitive decline due to aging. Therefore, it was found that it can be used not only as an agent for improving and treating memory and cognitive function, but also as a useful agent for preventing or treating various brain diseases including dementia.

[0152]

[0153] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. Containing SerpinA1e protein or a polynucleotide encoding the protein as an active ingredient, A pharmaceutical composition for the prevention or treatment of memory impairment, cognitive dysfunction or brain nerve disease.

2. In paragraph 1, A pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction or brain nerve disease, characterized in that the protein is composed of an amino acid sequence of sequence number 1.

3. In paragraph 1, A pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction or brain nerve disease, characterized in that the polynucleotide is composed of a base sequence of sequence number 2.

4. In paragraph 1, A pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction or neurological disease, characterized in that the SerpinA1e protein passes through the blood-brain barrier.

5. In paragraph 1, The above composition, Increased production of nerve cells; Increased expression of BDNF (brain-derived neurotrophic factor) protein; and A pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction or brain nerve disease, characterized by increasing the length and number of branches of nerve cells.

6. In paragraph 1, A pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction or a brain disease, characterized in that the brain disease is selected from the group consisting of mild cognitive impairment, Parkinson's disease, Huntington's disease, Alzheimer's disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy and frontotemporal dementia.

7. In paragraph 1, A pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction or brain nerve disease, characterized in that the SerpinA1e protein is a recombinant SerpinA1e protein to which a marker amino acid sequence of sequence number 7 for detection and purification of the protein is linked.

8. In paragraph 7, A pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction or brain nerve disease, characterized in that the recombinant SerpinA1e protein is composed of an amino acid sequence of sequence number 3.

9. In paragraph 8, A pharmaceutical composition for preventing or treating memory impairment, cognitive dysfunction or brain nerve disease, characterized in that the polynucleotide encoding the recombinant SerpinA1e protein comprises a base sequence of SEQ ID NO:

4.

10. A method for treating memory impairment, cognitive dysfunction or neurological disease, comprising a step of administering a recombinant expression vector containing SerpinA1e protein or a polynucleotide encoding the protein into a blood vessel of a mammal other than a human.

11. In paragraph 10, A method characterized in that the above protein consists of an amino acid sequence of sequence number 1.

12. In paragraph 10, A method characterized in that the above polynucleotide consists of a base sequence of sequence number 2.

13. In paragraph 10, The above SerpinA1e protein is, Crosses the blood-brain barrier; Increased production of nerve cells; Increased expression of BDNF (brain-derived neurotrophic factor) protein; and A method characterized by having an effect of preventing, improving or treating memory impairment, cognitive dysfunction or brain nerve disease by increasing the length and number of branches of nerve cells.

14. In paragraph 10, A method characterized in that the SerpinA1e protein is a recombinant SerpinA1e protein to which a marker amino acid sequence of sequence number 7 for detection and purification of the protein is linked.

15. In paragraph 14, A method characterized in that the recombinant SerpinA1e protein comprises an amino acid sequence of sequence number 3.

16. In paragraph 15, A method characterized in that the polynucleotide encoding the recombinant SerpinA1e protein comprises a base sequence of SEQ ID NO: 4.

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