Composition comprising hapln1 as active ingredient or preventing or treating senile degenerative brain diseases

Abstract

The present invention relates to a composition comprising HAPLN1 as an active ingredient for preventing or treating senile degenerative brain diseases. Specifically, recombinant human HAPLN1 protein (rhHAPLN1) lowers the protein level of p16 in cultured human astrocytes to inhibit cellular senescence caused by the accumulation of beta amyloid peptides, and further inhibits phosphorylation (p-p38 MAPK) of p38 MAPK protein, thereby also having the possibility of inhibiting inflammatory responses associated with the onset of Alzheimer's disease and Parkinson's disease. In addition, the recombinant human HAPLN1 protein (rhHAPLN1) exhibits significant memory and learning improvement effects in in vivo experiments performed using a mouse acute Alzheimer's disease model, and thus can be expected to exhibit preventive and therapeutic effects against Alzheimer's disease that may occur with aging and the like. In addition, the inhibitory effect of the rhHAPLN1 protein on cellular senescence and inflammatory responses of astrocytes can provide a very important clue for establishing prevention and treatment strategies not only for aging itself but also for brain functions, motor behaviors, memory, seizures, dementia, brain tumors, and the like.

Description

Composition for preventing or treating senile degenerative brain disease containing HAPLN1 as an active ingredient

[0001] The present invention relates to a composition for preventing or treating senile degenerative brain diseases, comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) as an active ingredient. Specifically, the present invention provides a composition for preventing or treating senile degenerative brain diseases such as Alzheimer's disease and Parkinson's disease, which are among the dementias that frequently occur in the elderly and are caused by extracellular accumulation of beta-amyloid peptides.

[0002] With the global aging trend, age-related central nervous system (CNS) diseases such as Alzheimer's and Parkinson's are rapidly increasing. However, no effective drugs have yet been developed to prevent or treat these diseases. Therefore, active research into these diseases is being conducted worldwide to meet the rapidly increasing medical demand. Notably, research on cellular senescence in non-neuronal cells such as astrocytes and microglia, which support the development of CNS diseases, has shifted from the traditional focus on neurons. This has led to the rapid discovery of new therapeutic concepts and related mechanisms.

[0003] Dementia is a disease that causes the loss of intellectual functions, such as thinking, memory, and reasoning, and has various known causes. Alzheimer's disease accounts for approximately 70% of dementia cases, followed by vascular and cerebral infarction-related dementia, which account for approximately 14%. The first and most common symptom experienced by Alzheimer's patients is a progressive decline in memory, a condition caused by brain abnormalities that can interfere with daily life. Early in the disease, patients experience difficulties registering, storing, and retrieving new information, especially short-term memories. As the disease progresses, even long-term memories acquired long ago become difficult to retrieve. When German physician Alois Alzheimer first described the disease in 1906, only a small number of people were diagnosed with it. However, it is now recognized as the most common cause of dementia, affecting 10% of those over 65 and nearly half of those over 85.

[0004] The exact pathogenesis and cause of Alzheimer's Disease (AD) are not clearly known, but it is caused by damage to brain cells, such as excessive production of harmful proteins called beta-amyloid peptides and their deposition in the extracellular space of brain tissue. This damage interferes with the ability of brain neurons to communicate, and it is caused by the formation of toxic beta-amyloid plaques (BAP) and tau protein tangles.

[0005] Meanwhile, Parkinson's disease is a degenerative disorder of specific brain regions, characterized by tremors at rest, stiffness due to increased muscle tone, slow voluntary movements, and postural instability due to difficulty maintaining balance. Many patients also exhibit cognitive impairment and dementia, symptoms similar to Alzheimer's disease. Its prevalence rate is approximately 1 in 100 people aged 65 and older. Like Alzheimer's, it shares the distinction of being a degenerative brain disease associated with age. However, looking at the clinical pathological characteristics of Parkinson's disease, unlike the hippocampus of Alzheimer's disease, it is caused by the formation of aggregates of alpha-synuclein protein in the substantia nigra pars compacta (SNpc) of the patient's midbrain and the resulting formation of Lewy bodies (abnormal protein conjugates), which kill dopaminergic neurons. It has also been revealed that this disease, like the mechanism of Alzheimer's disease, is caused by the clearance dysfunction of the Lewy bodies due to 'cellular senescence of astrocytes' that support dopaminergic neurons. Development of prevention or treatment for Parkinson's disease is actively underway, focusing on this.

[0006] Beta-amyloid is naturally produced in the body, but most of it is broken down and eliminated. However, some remains unbroken when inflammation occurs during the immune system's fight against invading microorganisms, leading to the degeneration of beta-amyloid peptides and the formation of plaques (B-amyloid plaques, BAP). These plaques accumulate and form between brain cells, disrupting normal brain function. Interestingly, research on the role of astrocytes (also known as astrocytes) in the brain and spinal cord in the formation and accumulation of these plaques has been actively conducted worldwide. Until now, astrocytes were known as a type of glial cell (or neuroglia) that auxiliaryly supports the normal function of neurons. Recently, it has been discovered that astrocytes, as the most numerous type of glial cell, play a major role in brain function through direct communication with neurons, and that they suppress the accumulation of beta-amyloid peptide secreted by neurons within tissues through endocytosis, preventing its accumulation in the extracellular space. Therefore, the role of astrocytes in the onset and progression of Alzheimer's disease is currently receiving significant attention worldwide.

[0007] Accordingly, the purpose of the present invention is to provide a composition for preventing, improving or treating a senile degenerative brain disease, which contains hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient.

[0008] The present invention provides a pharmaceutical composition for preventing or treating senile degenerative brain disease, which contains HAPLN1 or a gene encoding it as an active ingredient.

[0009] In addition, the present invention provides a health functional food composition for preventing or improving senile degenerative brain disease, which contains HAPLN1 or a gene encoding it as an active ingredient.

[0010] The present invention relates to a composition for preventing or treating age-related degenerative brain diseases, comprising HAPLN1 as an active ingredient. Specifically, recombinant human HAPLN1 protein (rhHAPLN1) suppresses cellular senescence due to accumulation of beta-amyloid peptide by lowering the protein level of p16 in cultured human astrocytes, and further suppresses the phosphorylation of p38 MAPK protein (p-p38 MAPK), thereby potentially suppressing the inflammatory response that occurs during the development of Alzheimer's disease and Parkinson's disease. In addition, in vivo experiments performed using a mouse acute Alzheimer's disease model showed significant memory and learning improvement effects, and thus, the composition can be expected to have a preventive and therapeutic effect on Alzheimer's disease that can be caused by old age, etc. In addition, the rhHAPLN1 protein's effect of suppressing cellular senescence and inflammatory response in astrocytes can provide very important clues for establishing prevention and treatment strategies for aging itself as well as brain function, motor behavior, memory, seizures, dementia, brain tumors, etc.

[0011] Figure 1 is a schematic diagram showing an experiment conducted by dividing mice into three groups by age group.

[0012] Figure 2 shows the results of measuring the expression of astrocyte markers and ECM molecules in the mouse cerebellar and cerebral cortices. The level of glial fibrillary acidic protein (GFAP), a specific marker of astrocyte senescence in mouse brain tissue, was higher in aged mice compared to young mice.

[0013] Figure 3 shows the results of measuring the expression of astrocyte markers and ECM molecules in the mouse cerebellar and cerebral cortices. Beta-amyloid plaques were higher in aged mice compared to young mice.

[0014] Figure 4 shows the results of measuring the expression of astrocytic markers and ECM molecules in the mouse cerebellar and cerebral cortices. Brevican, a proteoglycan that binds to the hyaluronic acid backbone by HAPLN1, HAPLN2, and HAPLN4, was found at higher levels in aged mice compared to young mice.

[0015] Figure 5 shows the results of measuring the expression of astrocyte markers and ECM molecules in the mouse cerebellar cortex and cerebral cortex. The expression level of HAPLN2 was higher in old mice compared to young mice.

[0016] Figure 6 shows the results of measuring the expression of astrocyte markers and ECM molecules in the mouse cerebellar and cerebral cortices. The expression level of HAPLN4 was also higher in aged mice compared to young mice.

[0017] Figure 7 shows the results of measuring the expression of astrocyte markers and ECM molecules in the mouse cerebellar and cerebral cortices. The expression level of p16, a cell-quiescent protein and marker of cellular senescence, was also higher in aged mice compared to young mice.

[0018] Figure 8 shows the results of an immunochemical quantitative method using antibodies to each marker to reconfirm the analysis results of each component and biomarker measured by a fluorescent staining method.

[0019] Figure 9 shows the results of comparing the mRNA expression levels of Versican, one of the lectican family members and its partner proteoglycan, in each age group, focusing on HAPLN1, the only protein whose level decreased in aged mice.

[0020] Figure 10 is a schematic diagram explaining the experimental background and basis of the present invention.

[0021] Figure 11 is Aβ1 -42 The effect of exogenous rhHAPLN1 on aging of astrocytes through reduction of p16 expression by .

[0022] Figure 12 shows the results confirming that the level of p38 phosphorylated (p-p38 MAPK) protein was significantly reduced in a dose-dependent manner by rhHAPLN1.

[0023] Figure 13 shows the results of evaluating the efficacy of memory and learning recovery using the Y-maze maze test and passive avoidance test as acute mouse models to prove the efficacy against Alzheimer's disease as an in vivo experiment.

[0024] Figure 14 shows the results of the pole test (A) and the rotarod test (B) for evaluating behavioral measures and motor function tests of Parkinson's disease (PD).

[0025] Herein, the present inventors completed the present invention by producing a novel recombinant HAPLN1 protein and confirming its effect of suppressing cell senescence and inflammatory response in astrocytes induced with beta-amyloid peptide as a pathogenic factor, and its effect of improving memory impairment and learning in a beta-amyloid peptide-induced mouse acute Alzheimer's disease model. Both Alzheimer's disease and Parkinson's disease are caused by aggregated proteins that are denatured by various factors and accumulate outside the cell as neurotoxic substances. That is, Alzheimer's disease is caused by the accumulation of aggregates of beta-amyloid peptide proteins, and Parkinson's disease is caused by the accumulation of aggregates of tau proteins. However, the location of the lesion and the uptake mechanism are different. That is, Alzheimer's disease mainly occurs in the hippocampus, which is the memory center, and Parkinson's disease mainly occurs in the substantia nigra, which is the dopaminergic nerve region in the center of the brainstem. Meanwhile, normal healthy astrocytes take up these different aggregated proteins into the cells. Beta-amyloid aggregates are taken up through endocytosis via LRP1, and alpha-synuclein protein aggregates are taken up through phagocytosis, thereby suppressing the accumulation of these neurotoxic substances. In this sense, both diseases can be said to have a common cause: astrocyte senescence caused by these protein aggregates. In other words, the common treatment strategy for both diseases is none other than suppressing astrocyte senescence.

[0026] Meanwhile, the inventors of the present invention have focused on the effectiveness of rhHAPLN1 in the acute mouse Alzheimer's disease (AD) model and the subacute Parkinson's disease (PD) model through various literature evidence and numerous research results using rhHAPLN1 protein, and to examine whether it is effective, they first cultured human astrocytes and added beta-amyloid peptide oligomers (oligomer Aβ1). - 42) to observe the cellular senescence response, and confirmed through analysis of several cellular senescence indicators, and observed that these indicators were significantly reduced by rhHAPLN1 treatment, securing the mechanistic basis for its effectiveness. As a result of performing the Y maze test and passive avoidance test, which are in vivo mouse Alzheimer's acute models, statistically significant cognitive and memory improvement effects were confirmed at each dose of 0.1 and 0.3 mg per kg of body weight, and statistically significant motor ability improvement effects were observed at a dose of 0.3 mg per kg of body weight through the stick test and rotarod test, which are Parkinson's disease models. In conclusion, rhHAPLN1 suggests the possibility as a preventive and therapeutic candidate drug substance effective for Alzheimer's disease at least through inhibition of cellular senescence of astrocytes.

[0027]

[0028] The present invention provides a pharmaceutical composition for preventing or treating senile degenerative brain disease, which contains HAPLN1 or a gene encoding it as an active ingredient.

[0029] Preferably, the HAPLN1 may be composed of an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

[0030] Preferably, the senile degenerative brain disease may be, but is not limited to, dementia, Alzheimer's disease or Parkinson's disease.

[0031] Preferably, the composition can reduce the level of p16 protein expression in astrocytes and inhibit phosphorylation of p38 MAPK protein (p-p38 MAPK), but is not limited thereto.

[0032]

[0033] The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants may include solubilizers such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and may include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.

[0034] The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of the active compound, etc. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Therefore, it can be adjusted according to various factors including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, body weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. For example, although not constant, it may be administered once or several times daily at a dose of generally 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg. The above dosage does not limit the scope of the present invention in any way.

[0035]

[0036] In addition, the present invention provides a health functional food composition for preventing or improving senile degenerative brain disease, which contains HAPLN1 or a gene encoding it as an active ingredient.

[0037] Preferably, the HAPLN1 may be composed of an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

[0038] Preferably, the senile degenerative brain disease may be, but is not limited to, dementia, Alzheimer's disease or Parkinson's disease.

[0039]

[0040] The health functional food composition of the present invention may be provided in the form of a powder, granules, tablets, capsules, syrup, or beverage. The health functional food composition may be used in combination with other foods or food additives in addition to the active ingredient, and may be appropriately used according to conventional methods. The amount of the active ingredient mixed may be appropriately determined depending on the intended use, for example, for preventive, health, or therapeutic treatment.

[0041] The effective dosage of the active ingredient contained in the above health functional food composition may be used in accordance with the effective dosage of the above pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range. It is certain that the active ingredient may be used in an amount exceeding the above range because there is no problem in terms of safety.

[0042] There are no special restrictions on the types of the above health foods, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0043]

[0044] The above "HAPLN1" is a protein that stabilizes hyaluronic acid by linking it to proteoglycan, and is a component protein within the extracellular matrix first discovered in the joints of vertebrates.

[0045] The “recombinant human HAPLN1 protein (rhHAPLN1 protein)” according to the present invention is a novel recombinant protein produced using the HAPLN1 protein.

[0046] As used herein, "prevention" refers to any action that inhibits or delays the onset of a senile degenerative brain disease, or at least one symptom of the disease, by administering a pharmaceutical composition or health functional food composition according to the present invention. It also includes treatment of a subject suffering from the disease to prevent or mitigate recurrence.

[0047] In this specification, “treatment” means any act of improving or beneficially altering the symptoms of a senile degenerative brain disease, or at least one symptom of the disease, such as alleviating, reducing, or eliminating the symptoms, by administering a pharmaceutical composition according to the present invention.

[0048] In this specification, “improvement” means any act of improving or beneficially changing the symptoms of a senile degenerative brain disease, or at least one symptom of the disease, such as alleviating, reducing, or eliminating the disease, by taking a health functional food composition according to the present invention.

[0049] In this specification, "pharmaceutical composition" means a composition administered for a specific purpose, and for the purposes of the present invention, it means administered to prevent or treat a senile degenerative brain disease, or at least one symptom of the disease.

[0050] In this specification, "health functional food" includes food manufactured and processed using raw materials or ingredients with functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means a food with high medical or healthcare effects processed to efficiently exhibit bioregulatory functions such as prevention or improvement of senile degenerative brain diseases, biodefense, immunity, and recovery, in addition to nutritional supply, for the purpose of the present invention.

[0051] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0052]

[0053] <Example>

[0054] 1. Amino acid sequence of recombinant human HAPLN1 protein (rhHAPLN1)

[0055] The amino acid sequence constituting the recombinant HAPLN1 protein is as follows.

[0056] DHLSDNYTLDHDRAIHIQAENGPHHLLVEAEQAKVFSHRGGNVTLPCKFYRDPTAFGSGIHKIRIKWTKLTSDYLKEVDVFVSMGYHKKTYGGYQGRVFLKGGSDSDASLVITDLTLEDYGRYKCEVIEGLEDDTVVVALDLQGVVFPYFPRLGRYNLNFHEAQQACLDQ DAVIASFDQLYDAWRGGLDWCNAGWLSDGSVQYPITKPREPCGGQNTVPGVRNYGFWDKDKSRYDVFCFTSNFNGRFYYLIHPTKLTYDEAVQACLNDGAQIAKVGQIFAAWKILGYDRCDAGWLADGSVRYPISRPRRRCSPTEAAVRFVGFPDKKHKLYGVYCFRAYN (SEQ ID NO: 1)

[0057]

[0058] 2. Expression, purification, and storage of recombinant human HAPLN1 protein (rhHAPLN1)

[0059] To produce recombinant human HAPLN1 (rhHAPLN1; gene number 2678736), a DNA vector encoding the amino acid sequence of recombinant human HAPLN1 was used as a host cell in Expi™ 293 cells (ThermoFisher Scientific Co., Waltham, Massachusetts USA). For purification, the vector was expressed with a secretory signal peptide, 10 histidines (H, His, histidine), and a TEV protease-recognition site inserted at the amino terminus. The culture medium was collected 3 days after vector infection and purified through a HisTrap column (GE Healthcare, IL, USA). The TEV protease-recognition sequence was cleaved with TEV protease, and molecules containing histidine were removed using DynaBeads (Thermo Fisher Scientific). The solution thus obtained was dialyzed against 40 mM Tris-HCl, 1 M NaCl, pH 8.0 for 16 hours. The protein concentration of the final purified product was 0.11 mg / ml, and the solvent was 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0, 50% glycerol. After aliquoting into single doses, it was stored in a -20℃ refrigerator for use.

[0060]

[0061] Because the HAPLN (hyaluronan and proteoglycan link protein) protein has a very high homology in amino acid sequence between humans and mice, the mice were divided into three groups by age group and the experiment was conducted (Fig. 1).

[0062]

[0063] Meanwhile, we measured extracellular matrix (ECM) components, cellular aging indicators, and beta-amyloid peptide accumulation in mice according to age. First, using a fluorescent staining method, the level of glial fibrillary acidic protein (GFAP), a specific marker of cellular aging in astrocytes in mouse brain tissue, was higher in old mice than in young mice. In other words, it can be seen that the level of astrocytic aging increases with age (Fig. 2).

[0064]

[0065] Beta-amyloid plaques were also higher in aged mice compared to young mice, suggesting that increased cellular senescence of astrocytes led to greater beta-amyloid accumulation in aged mice compared to young mice (Figure 3).

[0066]

[0067] Brevican, a type of proteoglycan, is known to be an important component of the perineuronal net (PNN), a special structure present in the extracellular matrix (ECM) of adult brain tissue, and is known to be a brain tissue-specific proteoglycan that increases long-term potentiation (LPT) in hippocampal CA1, but has no significant role in learning and memory. Brevican is a proteoglycan that binds to the hyaluronic acid backbone by HAPLN1, HAPLN2, and HAPLN4, and its level was higher in old mice than in young mice (Fig. 4).

[0068]

[0069] HAPLN2 expression levels were also higher in aged mice compared to young mice (Fig. 5). Currently, the role of HAPLN2 in brain function and the reason for this increase in levels with age are unknown.

[0070]

[0071] HAPLN4 expression levels were also higher in aged mice compared to young mice (Fig. 6). Currently, the role of HAPLN2 in brain function and the reason for its increased levels with age are unknown.

[0072]

[0073] p16, a cell-quiescent protein and marker of cellular senescence, was also expressed at higher levels in aged mice compared to young mice (Fig. 7). Thus, increased p16 protein expression in aged mice strongly suggests increased cellular senescence in various cells within brain tissue, including astrocytes.

[0074]

[0075] To reconfirm the analysis results of each component and biomarker measured by the above-mentioned fluorescent staining method, an immunochemical quantitative method using antibodies for each marker was investigated (Fig. 8). That is, the entire mouse brain tissue was homogenized, and the expression levels of each subtype of HAPLN protein (HAPLN1, HAPLN2, HAPLN4) were quantified and compared by age group using Western blotting analysis. As shown in Fig. 8C, the level of GFAP (glial fibrillary acidic protein), a marker of astrocytic senescence, increased with age. Similar to this increasing GFAP level, the expression levels of Brevican, Hapln4, and Hapln2 increased with age. Interestingly, however, as shown in the red border of Fig. 8B, the expression level of 'HAPLN1' decreased with age. These results are consistent with the results obtained by the present inventors through proteomics analysis of each serum protein after parabiosis between young and old mice. That is, a rejuvenating effect that converts an aging phenotype into a youthful phenotype is expected.

[0076]

[0077] Based on the above results, we compared the mRNA expression levels of HAPLN1, the only protein whose levels decreased in aged mice, and its partner proteoglycan, Versican, a member of the lectican family, across each age group (Fig. 9). As a result, both components were significantly reduced in middle-aged and old mice compared to young mice. Therefore, this suggests a role in the development of age-related degenerative diseases such as Alzheimer's disease and Parkinson's disease, which increase with age. We noted the age-related decline in HAPLN1 protein levels, along with the decline in its primary partner, Versican, and investigated whether this decline was related to the cellular senescence of astrocytes and whether such cellular senescence could be overcome by supplementation with HAPLN1 protein.

[0078]

[0079] Based on the results of previous experiments, Aβ1 -42 To determine whether oligomers induce cellular senescence in human astrocytes and whether increased p16 protein levels are reduced by direct supplementation of cultured astrocytes with recombinant human HAPLN1 (rhHAPLN1 protein), and further to determine whether Aβ1 in vivo experimental models -42 In a mouse model of Alzheimer's disease in which memory and learning impairment were induced by oligomer administration, we observed whether administration of rhHAPLN1 protein improved the memory and learning abilities of the induced mice. The figure below is a schematic diagram explaining the background and basis of this experiment (Fig. 10).

[0080]

[0081] As a result, as shown in Figure 11, Aβ1 in cultured astrocytes -42After 24 hours of treatment with rhHAPLN1 protein at various concentrations, the level of p16 protein was significantly reduced in proportion to the concentration of purified recombinant human HAPLN1 protein (rhHAPLN1). These results suggest that rhHAPLN1 protein may have reduced the level of p16 protein by suppressing its expression in astrocytes, which in turn suppressed cell cycle arrest and reversed cellular senescence.

[0082]

[0083] Meanwhile, the p38 family of protein kinase MAPKs has been shown to play an important role in the pathophysiology of the central nervous system, and its role in the cellular senescence of astrocytes has been particularly noted. It has been suggested that when beta-amyloid peptide stimulates astrocytes, p38 kinase activation leads to phosphorylation, which induces central nervous system inflammation through the secretion of SASP (senescence-associated secretory phenotype) such as reactive oxygen species (ROS) and IL-6, as well as cellular senescence due to increased expression of p16. Meanwhile, a similar mechanism involving p38 MAPK has been proposed as a mechanism of Parkinson's disease pathogenesis. Based on this literature evidence, cultured astrocytes were exposed to beta-amyloid peptide Aβ1. -42 After experimentally inducing phosphorylation of p38 protein by treatment, a decrease in such phosphorylation was observed upon dose-dependent treatment with rhHAPLN1.

[0084] As a result, as shown in Fig. 12, it was observed that the level of p38 phosphorylated form (p-p38 MAPK) protein was significantly reduced in a dose-dependent manner by rhHAPLN1. These results, along with the decrease in p16 level mentioned above, strongly suggest that rhHAPLN1 may suppress not only cellular senescence of astrocytes but also inflammatory response (CNS inflammation), one of the pathological causes of Alzheimer's disease and Parkinson's disease that appear throughout the central nervous system.

[0085]

[0086] Figure 13 shows that although various animal models have been established to date to prove efficacy against Alzheimer's disease through in vivo experiments, the most widely recognized and used model is an acute mouse model that evaluates the efficacy of memory and learning recovery using the Y-maze maze test and passive avoidance test.

[0087] Here is a description of the preparation and breeding of experimental animals for Fig. 13. That is, the experimental animals were 8-week-old female ICR male mice obtained from Young Bio Co., Ltd. (Seongnam-si, Gyeonggi-do), and were divided into a normal group (Normal group), a disease-induced group (Alzheimer group), and two doses (0.1 mg / kg rhHAPLN1 group and 0.3 mg / kg rhHAPLN1 group) of test substance administration group (rhHAPLN1). The feed was 5L79Rat / mouse and was fed ad libitum during the test period. Water was changed every other day and allowed to eat freely. Mice were placed in cages of 3-4 each, and the temperature and humidity in the breeding room were maintained at 22-24℃ and 50±5%, respectively, with a 12-hour day and night cycle. A Y-maze apparatus (San Diego Instruments Inc) and a passive avoidance apparatus (Ugo Basile 7551) were used, and beta-amyloid Aβ1 -42 The peptide used was Bachem (H1368).

[0088] Figures 13A and 13B show the results of the Y-maze maze test, and Figures 13C and 13D show the results of the passive avoidance test. Figures 13A and 13C are scatter plots, and Figures 13B and 13D are bar graphs. Seven male ICR mice weighing approximately 20 grams were used per group. In the normal group, saline (10 ml / kg) was administered intraperitoneally (ip) once a day for 7 days as an excipient for the test substance, and in the Alzheimer's disease-induced group, saline was administered ip once a day for 7 times, and aggregated Aβ was administered 1 day before the start of saline administration. 1-42 10 nM was administered by intracerebroventricular injection (icv), and the test substance 0.1 mg / kg rhHAPLN1 and 0.3 mg / kg rhHAPLN1 groups were administered once a day for 7 times, and aggregated Aβ1 was measured 1 day before the start of test substance administration. -42 10 nM (10% DMSO in PBS, used after 6 days of storage in a 37℃ incubator) was administered by intracerebroventricular injection (icv; intracerebral injection to bypass the blood-brain barrier BBB).

[0089] As a result, as shown in Figures 13A and 13B below, aggregated Aβ1 was used to induce inhibition of spontaneous alternation (Spontaneous alteration (%)) behavior of mice in the Y maze. -42The administration group (Alzheimer group) of (10 nM) showed statistically significant Alzheimer's disease induction compared to the normal group (Normal group) (p<0.001), and both the test substances 0.1 mg / kg rhHAPLN1 and 0.3 mg / kg rhHAPLN1 showed statistically significant increases compared to the suppressed spontaneous alternation rate of the induced disease group (Alzheimer group) (0.1 mg / kg administration group, p=0.0017; 0.3 mg / kg administration group, p=0.0072).

[0090] Meanwhile, as shown in Figures 13C and 13D below, aggregated Aβ1 for inducing the step-through latency time (sec) of the passive avoidance test -42 The administration group (Alzheimer group) of (10 nM) showed a statistically significant decrease in retention time, indicating that Alzheimer's disease was induced (p<0.0029). The decreased retention time was increased by administration of 0.1 mg / kg rhHAPLN1 of the test substance, but did not show statistical significance. However, administration of 0.3 mg / kg rhHAPLN1 showed a statistically significant increase compared to the suppressed retention time (Step-through latency time (sec)) of the induced disease group (Alzheimer group) (0.1 mg / kg, NS; 0.3 mg / kg, p=0.0300).

[0091]

[0092] These are the results of the pole test (Fig. 14A) and the rotarod test (Fig. 14B), which were performed based on the subacute model established by Liebetanz et al. to evaluate behavioral measures and motor function tests of Parkinson's disease (PD). Eight male C57BL / 6 mice (26–32 g, Orient Bio, Gyeonggi-do) weighing approximately 30 g were used per group. Physiological saline was used as a solvent for the disease-inducing substance MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and the efficacy test substance rhHAPLN1. The normal group was administered physiological saline (10 ml / kg) by intraperitoneal injection (ip) once a day for 5 days, and the PD disease-inducing group was administered 30 mg (10 mg / ml saline solution, stored at 4℃) of the PD-inducing substance MPTP (Sigma-Aldrich, St. Louis, MO, USA) per kg of body weight by intraperitoneal injection (ip) once a day for 5 days. Meanwhile, the test substance groups of 0.1 mg / kg rhHAPLN1 and 0.3 mg / kg rhHAPLN1 were administered intracerebroventricular injection (i.c.v.; injection to bypass the blood-brain barrier BBB) once a day for 5 days, and on the 6th day, the rod test and rotarod test for PD test evaluation were performed. Figure 14A shows that the total time it takes for the mice to descend from the upper end of the rod (height 55 cm, diameter 1 cm) to the floor was measured three times and averaged to evaluate the motor behavioral ability of the experimental animals.

[0093] Compared to the normal group of 20.9 seconds, the PD-induced group showed a significant delay in average time of 64.2 seconds (p < 0.0001), indicating that the PD disease model was induced. The group administered rhHAPLN1 (0.1 mg / kg) showed a marked decrease trend of 55.7 seconds compared to the PD group, but there was no statistical significance (p = 0.0656). However, the group administered 3 times the dose of rhHAPLN (0.3 mg / kg) showed a statistically significant decrease of 44.4 seconds (p = 0.0011). Meanwhile, Figure 14B shows the results of the rotarod test conducted to evaluate motor function. While the normal group stayed on the rotating rod for 341.7 seconds, the PD group recorded 73.6 seconds, confirming a significant decrease in motor function as they fell significantly faster than the normal group (p = 0.0001). In this exercise test model, the rhHAPLN1 (0.1 mg / kg) group showed a significant increase of approximately 2.1 times compared to the PD group (p = 0.0002) to 157.5 seconds, and the group administered rhHAPLN1 (0.3 mg / kg) showed a significant increase of approximately 2.7 times compared to the PD group (p < 0.0001) to 196.4 seconds, showing statistically significant improvement in motor function in both doses.

[0094]

[0095] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating senile degenerative brain disease, comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient.

2. A pharmaceutical composition according to claim 1, characterized in that the HAPLN1 comprises an amino acid sequence represented by sequence number 1.

3. A pharmaceutical composition according to claim 1, characterized in that the senile degenerative brain disease is dementia, Alzheimer's disease, or Parkinson's disease.

4. A pharmaceutical composition according to any one of claims 1 to 3, characterized in that the composition reduces the level of p16 protein expression in astrocytes and inhibits phosphorylation of p38 MAPK protein (p-p38 MAPK).

5. A health functional food composition for preventing or improving senile degenerative brain disease, containing HAPLAN1 or a gene encoding it as an active ingredient.

6. A health functional food composition according to claim 5, wherein the HAPLN1 is composed of an amino acid sequence represented by sequence number 1.

7. A health functional food composition according to claim 5, wherein the senile degenerative brain disease is dementia, Alzheimer's disease, or Parkinson's disease.

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