KR20170085792A - Compositions containing Thrombospondin-1 for preventing or treating Alzheimer's disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of Alzheimer's disease, which comprises a thrombospondin-1 (TSP-1) protein or a gene thereof as an active ingredient. The composition of the present invention inhibits amyloid beta toxicity Dendritic spine loss can be prevented or restored.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating Alzheimer's disease comprising TSP-1 (Compositions containing thrombospondin-1 for preventing or treating Alzheimer's disease)

The present invention relates to a composition for preventing and treating Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia and is a representative neurodegenerative disease. It is estimated that more than 20% of elderly people over 80 years old are affected by Alzheimer 's disease.

Alzheimer's disease is characterized by the formation of senile plaques in which amyloid precursor protein (APP) is sequentially cleaved by β, γ-secretase and β-amyloid (Aβ peptide) The neurofibrillary tangle (NTF) due to hyperphosphorylation of the Tau protein, a vascular-associated protein, is a major pathological feature.

Fundamental treatment methods for Alzheimer's disease have not yet been developed, and medications that can relieve symptoms and delay progression have been used in clinical settings. As a representative drug, an acetylcholinesterase inhibitor is used in early Alzheimer's disease, which can not prevent progression of the disease and slows the progression of the disease by half a year to two years. In moderate-to-advanced Alzheimer's disease, NMDA receptor antagonist Is used.

However, various changes in the brain tissue of patients with Alzheimer's disease (AD) are caused by environmental factors and various etiological factors, and thus, there is no definite diagnostic method and treatment method.

Korean Patent No. 10-1114800 discloses a composition for preventing or treating neurological diseases including mesenchymal stem cells or a culture thereof.

The present invention provides a composition for preventing and treating Alzheimer's disease.

In one embodiment, the present invention relates to a pharmaceutical composition for preventing or treating Alzheimer's disease comprising TSP-1 (Thrombospondin-1) protein or a gene thereof as an active ingredient.

In particular, the TSP-1 protein or gene according to the present invention can prevent the loss of dendritic spines due to amyloid beta or amyloid plaques comprising it.

In particular, the TSP-1 protein or gene according to the present invention may alleviate or ameliorate a change in synaptic protein expression or a decrease in activity by amyloid beta or an amyloid plaque comprising the same.

In another embodiment, the present application provides a neuronal intracellular CACNA2D1 (calcium channel, voltage-dependent, alpha 2 / delta subunit 1) regulatory kit in an invivo or Invitro containing TSP-1 (Thrombospondin- do.

In another embodiment, there is provided a kit for alleviating or ameliorating a synaptic protein expression change or activity reduction by amyloid beta in neurons in an invivo or an invitro including a TSP-1 (Thrombospondin-1) protein or a gene thereof.

In another aspect, the present invention provides a method of treating a neuronal cell damaged by an amyloid beta with a TSP-1 (Thrombospondin-1) protein or a gene thereof in an in vivo or in vitro manner, -dependent, alpha 2 / delta subunit 1).

In another embodiment, there is provided a method of treating a neural cell damaged by dendritic cells with amyloid beta, comprising the step of treating a TSP-1 (Thrombospondin-1) protein or a gene thereof with an amyloid beta-induced synaptic protein Lt; RTI ID = 0.0 > alleviation or < / RTI >

The composition according to the present invention is effective for the prevention and treatment of Alzheimer's disease. The composition according to the present invention can be effectively used for the treatment or prevention of Alzheimer's disease by alleviating or preventing loss of dendritic spines due to decrease of synaptic protein activity due to changes in synaptic protein expression by amyloid beta (Aβ) in the brain.

Figure 1 shows that the expression of TSP-1 in the brain of AD patients and AD model mice was altered. (A) shows information on the normal control group and AD patients used in the present experiment. (B) is the quantification of TSP-1 levels in the brain of AD patients (n = 6 per group) compared to the normal control. (C) shows the level of each TSP-1, TSP-2, and synaptic proteins (PSD-95 and synaptophenic) obtained from the hippocampus obtained from the hippocampus of Alzheimer's animal models Tg6799 and littermate control mice (Control group; n = 5 per group). The lower panel shows the quantization data for the WB bands. * p < 0.05, ** p < 0.01 for litterate control mice. (D) TSP-1 expression levels in the cortex of Tg 2576 and littermate control mice. GAPDH served as a control group (n = 4, ** P <0.01 per group). Abbreviations: AD, Alzheimer's disease; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ns, nonsignificant; PSD-95, post synaptic density protein 95; TSP, Trombospondin; WB, Western Blot.
Figure 2 shows that A [beta] decreases TSP-1 release from astrocyte. (A) WB analysis of TSP-1 levels in U373MG astrocytoma cell medium. H ₂ O ₂ was used as a negative control for TSP-1 secretion, and insulin was treated to increase TSP-1 secretion. Blat represents at least three independent experiments. (B) The level of TSP-1 secreted in U373MG was quantified by ELISA; rev-Aβ shows an inverted form of Aβ _{1 -42.} Data were obtained at least three times per group (N = 3 experiments). Data are expressed as mean SEM. * P &lt;0.05; ** For P <0.01 vehicle- (dimethylsulfoxide) treated cells. (C) WB analysis of TSP-1 secreted from the first astrocytic cells. Representative images are shown (N = # experiment). ** P <0.01, *** P <0.001 compared to vehicle-treated cells. (D) Time-dependent changes in cell and secreted TSP-1 levels by Aβ in primary astrocytes (N = 4 experiment). * p < 0.05, ** p < 0.01, *** p < 0.001 for vehicle treated cells (in medium); P < 0.05 (in cells) for vehicle treated cells. (E) WB analysis of TSP-2 in medium (N = 3 experiment) under Aβ treatment conditions. (F) TSP-1 level changes after treatment with 2 mM oligomer A [beta] or fibrous A [beta]. The figures are mean SEM. Representative image is shown (N = 3 experiment). ** P < 0.01, *** P < 0.001. Abbreviations: Aβ, amyloid beta; ELISA, enzyme immunoassay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SEM, mean standard error; TSP, Trombospondin; WB, Western Blot.
Figure 3 shows that decreasing A [beta] -induced TSP-1 secretion is mediated by autophagy activation. (A) The transcription level of TSP-1 was determined using a promoter reporter assay. (B) RT-PCR analysis of TSP-1 under A [beta] -treated conditions. (C) Aβ-inducible autophagosome accumulation in U373MG astrocytes. (D) Based on the RFP-LC3 signal, Aβ effects on autogenous somatogenesis. The cells were transfected with the RFP-LC3 fusion construct. The scale bar is expressed as 10 mu m. Representative image is shown (N = 3 experiment). (E) TSP-1 levels secreted from U373MG astrocytes after treatment with several inhibitors and activators in the presence of A [beta] were quantified by ELISA. The data were obtained at least three iterations per group. ** P < 0.01, for vehicle-treated cells; # P <0.05 and ## P <0.01, for cells treated with Aβ. (F) WB analysis of TSP-1 level and intracellular beclin 1 level in media. beta -actin served as a loading control. *** P < 0.001 for vehicle-treated cells; ## P <0.01, for cells treated with Aβ. (G) 3D-SIM reconstructed images of TSP-1 expression in autopagosomes. The green signal indicates LC3 and the red signal indicates TSP-1. 3D-SIM images were taken in the Z-direction with a thickness of 0.150μm and reconstructed into 3D-volume images. The scale bar represents 2 m. Abbreviations: Aβ, amyloid beta; Baf, Bartholomycin; CG, chloroquine; ELISA, enzyme immunoassay; KD, knockdown; RFP-LC3, red fluorescent protein-microtubule-associated protein 1A / 1B-light chain 3; RT-PCR, RT-PCR; SIM, super-resolution microscope; TSP, Trombospondin; WB, Western Blot.
Figure 4 shows that TSP-1 prevents the loss of A [beta] -mediated dendritic spines and loss of functional synaptic activity by A [beta] in primary hippocampal neurons. (A) Anti-TSP-1 IgG is used to show that endogenous TSP-1 is depleted in ACM. (B) the role of endogenous TSP-1 on A [beta] -induced synaptic loss. Data were obtained at least three times per group (N = 3 experiments). Data are expressed as mean SEM. ** p < 0.01, vehicle-treated cells compared; #p <0.05, ## p <0.01, vehicle-treated ACM-cultured cells; P P < 0.05, relative to A [beta] -treated normal IgG-depleted ACM cultured cells. (C) synaptic protein, NR2A, and PSD-95. * P < 0.05, contrast to vehicle-treated cells; #P < 0.05, compared to cells treated with A [beta]. (D) The effect of TSP-1 on Aβ-mediated dendritic spine loss in cultured primary rat hippocampal neurons using immunocytochemistry. The red signal represents MAP2B for microtubule staining and the green signal represents vGluT1 for excitatory synaptic measurements. Synaptic concentrations were analyzed by counting the red signal (vGluT1-positive dendritic spines) using ImageJ and expressed every 1m of apical dendrite. Scale bar, 2μm. * P < 0.05, vehicle-treated cells; ## P <0.01, relative to Aβ-treated cells. (E) FM4-64 staining of functional pre-synaptic terminals in neurons treated with Aβ and / or TSP-1. The right panel shows a quantitative analysis of changes in mean at the FM4-64 puncta intensity. Scale bar, 20μm. *** P < 0.001, compared to vehicle-treated cells; ### P <0.001, relative to Aβ-treated cells. Abbreviations: ACM, astrocytic-conditioned medium; A [beta], amyloid beta; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAP2B, microtubule-associated protein 2B; NR2A, NMDA receptor 2A; PSD-95, post synaptic density protein 95; SEM, mean standard error; TSP, Trombospondin; vGluT1, vesicle glutamate transporter 1.
Figure 5 shows that TSP-1 restores A [beta] -induced degeneration in synaptic protein expression in invivo. (A) Indicates recovery of A [beta] -induced synaptic loss in the ipsilateral hippocampal subiculum in which the TSP-1 protein is present. Expression levels of synaptic proteins were analyzed in melts prepared from the ipsilateral and contralateral hippocampal transition regions of Tg6799 and the rat matte control mice in which the TSP-1 protein was present. (B) The data of FIG. 5A is quantified. * p < 0.05, ** p < 0.01, versus hippocampal migration of littermate control mice; # P < 0.05, compared to the contralateral hippocampal migration of Tg6799 mice. Abbreviations: Aβ, amyloid beta; Contra, contralateral; Ips, ipsilateral; LT, littermate control mice; NR1, neurorein-1; NR2A, NMDA receptor 2A; PSD-95, post-synaptic density protein 95; SNP, synaptophysin; Tg, Tg6799; TSP, Trombospondin.
Figure 6 shows that TSP-1 restores A [beta] -inducedly reduced synaptic protein through the [alpha] 2 delta receptor. (A) In? 2? 1 KD cells, PSD-95 expression levels were determined as WB under Aβ and / or TSP-1 treated conditions. The right panel shows quantitative data. Data were obtained at least three times per group (N = 3 experiments). Data are expressed as mean SEM. * p < 0.05, vehicle-treated cells; #p < 0.05, relative to A [beta] -treated cells. (B) The effect of TSP-1 and its receptor (? 2? 1) on Aβ-mediated dendritic spine loss in cultured primary rat hippocampal neurons using phalloidin staining. The lower panel is an enlarged image of the area indicated by the red line, and the arrowhead represents the dendrite spine. Scale bar, 20μm. (C) Quantitative analysis of FIG. 6B. ** P < 0.01, vehicle-treated cells; # P < 0.05, relative to A [beta] -treated cells. (D) and CD47 (E), which are known TSP-1 receptors, by the TSP-1 (D and E) Aβ-induced dendritic spine loss recovery by TSP-1. Data were obtained three times (N = 3 experiments). Data are expressed as mean SEM. * p &lt; 0.05, for vehicle-treated cells; # p &lt; 0.05, for A [beta] treated cells. Abbreviations: Aβ, amyloid beta; alpha 2 delta-1, alpha-2-delta-1; CD47, differentiation cluster 47; KD, knockdown; NL1, &lt; / RTI &gt; SEM, mean standard error; PSD-95, post-synaptic density protein 95; TSP, Trombospondin; WB, Western Blot.
Figure 7 is a graphical representation of A [beta] leading to a decrease in TSP-1 levels secreted in astrocytes in an autophagy-dependent manner. Co-culture of TSP-1 in the presence of Aβ (endogenous and / or exogenous) restored Aβ-induced synaptic pathology such as dendritic spine density reduction and synaptic activity damage through the neuronal TSP-1 receptor α2δ1. These data show that TSP-1 is a potent therapeutic target for the onset of Alzheimer's, particularly for synaptic dysfunction. Abbreviations: AD, Alzheimer's disease; A [beta], amyloid beta; alpha 2 delta-1, alpha-2-delta-1; TSP, Trombospondin.

The present invention is based on the discovery that thrombospondin-1 can be used in the treatment of dementia by preventing the loss of dendritic spines by alleviating or restoring the expression and activity of synaptic protein expression and activity by amyloid plaques comprising amyloid beta .

Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for preventing or treating Alzheimer's disease, which comprises, as an active ingredient, a thrombospondin-1 (TSP-1) protein or a gene thereof.

The term &quot; Thrombospondin-1 (TSP-1) &quot; as used herein refers to a protein encoded by the THBS1 gene as a member of the thrombospondin family. TSP-1 is a disulfide bonded homotrimer protein, an adhesion glycoprotein that mediates intracellular or cell-to-matrix interaction, and is known to interact with at least 12 cell adhesion receptors. Plasma aggregation, Production, and tumorigenesis.

In the brain of Alzheimer's patients, Aβ causes alteration of synaptic protein expression and loss of synaptic protein activity resulting in loss of dendritic spines. TSP-1 according to the present invention prevents or ameliorates a decrease or loss of A? In the brain of a patient with Alzheimer's disease. Although not limited to the mechanism according to the present invention, the TSP-1 according to the present invention is useful for alleviating synaptic protein loss through the α2δ1 receptor, which is one of the TSP-1 receptors, It can be useful for preventing or treating diseases.

Therefore, for the purpose of achieving the object of the present invention, various origins and / or forms of TSP-1 gene and / or protein may be used as long as the effect of the present invention is attained. A gene in which a part of the base sequence is artificially modified so as to favor characteristics such as expression in a cell or stability of the protein as well as a wild type sequence and a gene in which a part of the naturally found base sequence is modified or a fragment thereof . Modifications of the gene sequence may or may not involve modification of the corresponding amino acid, and in the case of accompanied by modification of the amino acid, the gene in which such modification occurs is one in which at least one amino acid in the encoded protein is substituted, deleted, And / or an inserted amino acid sequence, which comprises mutants, derivatives, alleles, variants and homologues thereof. In the case where the mutation of the gene sequence does not involve the modification of the amino acid in the protein, for example, there is a mutation in the axis, and such degeneracy mutants are also included in the gene of the present invention.

Modifications of artificial gene sequences can be performed by methods well known to those skilled in the art, for example, site directed mutagenesis (Kramer et al, 1987), error induced PCR (Cadwell, RC and GF Joyce. Appl., 2: 28-33.), Point mutation method (Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd Ed. 2001, Cold Spring Harbor Laboratory Press). In one embodiment according to the present invention, the gene described above may be provided in the form of a vector operably linked to a promoter so as to be capable of expression in the cell of the subject to which the pharmaceutical composition is administered. Proteins used herein can be prepared using methods known in the art.

In one embodiment, the method of producing a protein is by using a gene recombination technique. For example, a vector containing the corresponding gene encoding the protein may be transferred to a prokaryotic or eukaryotic cell such as an insect cell or a mammalian cell for expression, followed by purification. The plasmid can be used, for example, by cloning the gene into an expression vector such as pET28b (Novagen), transferring the gene to the cell line, and then purifying the expressed protein, but is not limited thereto. The synthesized protein was purified by column chromatography including precipitation, dialysis, ion exchange chromatography, gel-permeation chromatography, HPLC, reversed-phase HPLC, SDS-PAGE for free, and affinity column using anti-screening protein antibody Can be separated and purified.

In one embodiment according to the present application, the TSP-1 gene and protein are derived from mammals such as apes, humans and the like, and are particularly human. The TSP-1 gene and the protein may also be used as long as they achieve their full length or effect of the present invention. In one embodiment according to the present application, the full-length TSP-1 gene and / or protein is used. In one embodiment according to the present application, mammalian, in particular human, TSP-1 is used and this protein sequence is searchable under Gene ID: 7057 (www. Ncbi.nlm.nih.gov/gene/7057).

As used herein, the term "Alzheimer &apos; s disease &quot; refers to a degenerative brain disease that causes dementia and gradually develops into a disease in which cognitive function including memory is progressively worsened. Alzheimer's disease, Alzheimer's disease, and familial Alzheimer's disease (FAD), all of which occur at age 65 or older. Most pre-emergent Alzheimer's disease is caused by a unique gene mutation, and familial Alzheimer's disease is caused by genetic mutations that cause some well-known Alzheimer's disease. Since TSP-1 according to the present invention has a mechanism to mitigate the toxic effects of amyloid beta, it can be used for the prevention and treatment of various kinds of Alzheimer's disease caused by amyloid beta.

As used herein, "amyloid plaque" may be an insoluble fibrous protein aggregate comprising amyloid beta. The amyloid plaque may be present in a cell, on a cell surface, and / or in a space between cells. For example, it may be in a space between cells of a neural tissue.

As used herein, "nerve tissue" includes central nervous system tissue, such as the brain. Brain tissue may include cerebral, cerebellar, and hippocampal tissues. The cerebral tissue contains the cerebral cortex. The nerve tissue contains not only the nerve tissue itself but also the nerve cell, and the nerve cell is one of the constituent elements of the nerve tissue. Such neurons include neuronal cells and / or microglial cells. Culturing of neural tissue involves culturing neurons, such as neurons and / or microglia cells, in vitro or in vivo. Inhibitory culture includes culturing cells in an individual by administering the cells to the individual.

As used herein, the term "treatment" refers to any action that improves or alters the relevant symptoms by administration of a composition according to the invention. Those skilled in the art will be able to ascertain, by reference to the data provided by the Korean Medical Association, the precise criteria of the disease for which the composition of the present invention is effective, .

The term "prophylactic," as used herein, refers to any act that inhibits or delays the onset of a related disorder upon administration of a composition according to the present disclosure. Neuropsychological tests can be used to diagnose patients with dementia with mild cognitive impairment. An average of 12% of patients with mild cognitive impairment develops into Alzheimer's disease for one year, leaving mild cognitive impairment unchanged Administration of the composition of the present invention which can alleviate or reduce the toxicity by amyloid plaques comprising amyloid beta can prevent or progress the progression of Alzheimer's disease since about 80% You will see a slowing effect.

Accordingly, the composition of the present invention can be produced by a pharmaceutical composition. The pharmaceutical compositions may be administered simultaneously or sequentially and may be used alone or in combination with other pharmaceutical active ingredients and methods of using biological response modifiers for surgery, treatment of such diseases.

The therapeutic agent or pharmaceutical composition according to the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. &Quot; Pharmaceutically acceptable &quot; refers to compositions which are physiologically acceptable and which, when administered to humans, do not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or the like. Examples of pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, aqueous carriers for parenteral administration such as aqueous glucose and glycols, etc., and may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the composition according to the present invention may contain various additives such as a suspending agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, an interface activator, a diluent, an excipient, a pH adjuster, An antioxidant, and the like. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington ' s Pharmaceutical Sciences, Current Edition.

The composition of the present invention may be prepared in a unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Into a capacity container. The formulations may be in the form of solutions, suspensions or emulsions in oil or aqueous media, or in the form of powders, granules, tablets or capsules.

The method of administration of the pharmaceutical composition of the present invention can be easily selected according to the formulation, and can be administered to mammals such as livestock, human, and the like in various routes. For example, it may be formulated in the form of powders, tablets, pills, granules, dragees, hard or soft capsules, liquids, emulsions, suspensions, syrups, elixirs, external preparations, suppositories, sterilized injection solutions, Or parenterally, and parenteral administration may be particularly preferable.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and parenteral administration is particularly preferred. The dose varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and time, but can be appropriately selected by those skilled in the art. In one embodiment according to the present invention, the composition is administered topically to the bone marrow and around the physically and / or pathologically damaged bone.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically or therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type, severity, The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, 60 kg), it is about 1 ng to 10 mg / day, especially about 1 μg to 1 mg / day. It will be apparent to those skilled in the art that doses may be additive or subtracted, as the dosage can vary depending on various conditions, and thus the dose is not intended to limit the scope of the invention in any way.

The number of administrations can be administered once or several times a day within a desired range, and the administration period is not particularly limited.

The TSP-1 protein or a gene thereof according to the present invention can be used to treat or prevent amyloid beta toxicity &lt; RTI ID = 0.0 &gt; (I) &lt; / RTI &gt; through &lt; RTI ID = 0.0 &gt; mitochondrial &lt; / RTI &gt; Such as damage to dendritic nerves caused by nerve cells or tissues can be suppressed.

In this respect, the present invention also relates to a method of inhibiting damage to nerve cells or tissues caused by amyloid plaque toxicity including amyloid beta by the TSP-1 (Thrombospondin-1) protein or its gene in an in vivo or in vitro manner, CACNA2D1 -dependent, alpha 2 / delta subunit 1) modulation, alteration of synaptic protein expression by amyloid beta in neurons or mitigation or restoration of activity.

Thus, in one embodiment, the present invention provides a kit for regulating neuronal or neuronal CACNA2D1 (calcium channel, voltage-dependent, alpha 2 / delta subunit 1) comprising TSP-1 (Thrombospondin- The nerve cell provides an intracellular CACNA2D1 regulatory kit in which the dendritic cells are damaged cells by amyloid beta.

In another embodiment, there is provided a kit for alleviating or ameliorating a synaptic protein expression change or activity reduction by amyloid beta in a neuron comprising a TSP-1 (Thrombospondin-1) protein or a gene thereof.

In another aspect, there is provided a method of treating a neural cell damaged by an amyloid beta with a TSP-1 (Thrombospondin-1) protein or a gene thereof in an in vivo or in vitro manner, -dependent, alpha 2 / delta subunit 1).

A synaptic protein expression change or activity by amyloid beta of a neuron in an in vivo or in vitro manner, comprising the step of treating the neuron with damage to the dendritic cells by amyloid beta, or a TSP-1 (Thrombospondin-1) Provides a mitigation or recovery method.

The protein or gene contained in the kit according to the present invention and used in the treatment of the method may be referred to above.

Embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Materials and Methods

Animals and tissues

(Hsiao et al., 1996, Science 274, 99-102), Tg6799 (2, 6, and 9 months of age, respectively) = 5) (Oakley et al., 2006, J Neurosci. 25, 2702-2711) and nontransgenic littermate control mice (B6SJL) were used in the experiments. The Tg6799 mouse (formerly known as JAX stock no. 008730) is a member of the family PSA harboring 2 family AD (FAD) mutations (M146L and L286V) as well as the Swedish (K670N, M671L), Florida (I716V) FAD) mutants are also overexpressed in mutant human APP695. All mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and processed and stored at the National University mouse facility. All experiments were approved by Seoul National University's Laboratory Animal Resources Service. Post-human brain tissue was obtained from Dr. Liu Hoon (Boston University, Boston, Mass., USA) and the information is provided in FIG. 1A.

orientation  Injection( Stereotaxic  injection)

The mice were anesthetized (in 1 mL / kg intraperitoneal) with a solylate and lumped mixture and placed in a stereotaxic frame (myNeuroLab, St. Louis, Mo., USA). TSP-1 was dissolved in phosphate buffered saline (PBS). According to the Bregma landmark adopted by Paxinos and Watson atlas, 3 ml of saline or TSP-1 (0.5 mM) was injected into the hippocampal subiculum (4.16 mm AP, 3.25 mmML, and 4.0 mm DV) (unilaterally). Animal processing and management were approved by the Animal Experimentation Ethics Committee of Seoul National University.

Cell culture and drug treatment

Human astrocytoma U373MG cells (ATCC number: HTB-17) were cultured in RPMI 1640 containing 10% fetal bovine serum and 0.1 mg / mL penicillin and streptomycin (P / S; Sigma-Aldrich, St. Louis, Mo., USA) Lt; RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt; in humidified 5% CO ₂ air. The U373MG cells (5x10 ⁵ cells) were placed in a 6-well plate. The primary hippocampal neurons were prepared in E18 Sprague-Dawaur rats embryos according to the slightly modified method of the existing method (Brewer et al., 1993, J. Neurosci. Res. 35, 567-576). The neurobasal supplemented with B27 supplement (Invitrogen, Carlsbad, CA, USA), L-glutamine (0.5 mM), and 0.1 mg / mL P / S (Sigma- Aldrich) Experiments were performed in media for 19-21 days in Invitro (DIV). According to the conventional method (Jung et al., 2012, J. Neurosci. 32, 3081-3087), primary astrocytes were prepared from neonatal (P1) imprinted control region mice. The astrocytes were subjected to 2 passages in the experiment. The cells were treated with 500 ng / mL of human recombinant TSP-1 (R &amp; D systems, Minneapolis, MN, USA) alone or co-treated with Aβ and incubated for 24 hours. Small interfering RNA (RNA) and short hairpin RNA are available from Bioneer Inc. Or SantaCruz Biotechnology (CA, USA) and transfected using RNAimax or Lipofectamine 2000 (Invitrogen) according to the manual. Drugs used in this experiment were as follows: insulin (100 nM), H ₂ O ₂ (50 μM), 3-methyladenine (2 mM), bar philomicin (10 nM), chloroqueen (100 μM), TNF- 100 mu M), and rapamycin (100 nM) from Sigma-Aldrich; GM6001 (5 [mu] M) from Calbiochem (San Diego, CA, USA).

Western Blat  analysis

Harvesting cell pellets and brain melts were prepared according to the conventional method (Son et al., 2014, Cell Death Dis. 5, e1188). Anti-N-methyl-D-aspartate receptor 2A and anti-NR1 (1: 2000; Millipore, Schwalbach, Germany) were used for Western blot analysis. Anti-cAMP response element-binding protein (CREB) and anti-p-CREB (S133) (1: 2000, Cell Signaling Technology, Beverly, MA, USA); Anti-microtubule-associated protein 1A / 1B-light chain 3 (1: 2000; MBL; Cell Signaling Technology); Anti-α-actinin and anti-a2d1 (1: 1000; SantaCruz); Anti-TSP-1, anti-TSP-2, anti-CD47, anti-post synaptic density protein 95 (anti-PSD-95) and anti-beclin 1 (1: 2000; Abcam, Cambridge, MA, USA); Anti-SNP, anti-beta-actin, anti-alpha-tubulin, and anti-glyceride 3 phosphate dehydrogenase (1: 4000; Sigma-Aldrich). The immune response was determined by chemiluminescence (GE Healthcare, Piscataway, NJ, USA). The chemiluminescent signal was detected with a digital image analyzer (LAS-3000; Fuji Film Inc., Tokyo, Japan).

Aβ (Amyloid beta) production

Aβ was prepared according to the conventional method (Cha et al., 2012, PLoS One 7, e34929; Son et al., 2012, Neurobiol. Aging, 1006.e11-1006.e23). Most of the Aβ forms used were oligomers, but some monomers were also present in the mixture. To produce fibrous A [beta], 10 [mu] M HCl was added to make hexafluoroisopropanol-dissolved A [beta] in a final concentration of 100 [mu] M and the peptide was incubated at 37 [deg.] C for 24 hours, J. Biol. Chem. 277, 32046-32053).

TSP -1 emission measurement

Extracellular TSP-1 in the culture medium of U373MG cells was determined using a trichloroacetic acid (TCA) or TSP-1 ELISA kit (DTSP-10, R & D systems) according to the manual. To analyze the protein in the medium, the present inventor performed TCA precipitation according to the existing method (Son et al., 2012, Diabetes 61, 3126-3138). In summary, cell culture medium was centrifuged at 5000 rpm for 5 minutes to remove cell debris to allow for TCA uptake (up to 10%).

LC3-II Translocation (translocation) experiment

To analyze the red fluorescent protein (RFP) -LC3, a plasmid encoding RFP-LC3 was transfected into U373MG cells. LC3 puncta was visualized with a confocal laser scanning microscope (FV10i-w, Olympus, Japan).

RNA isolation and Reverse transcription  Polymerase reaction

Total RNA was extracted using TRIzol reagent in U373MG cells treated with vehicle or Aβ (Gibco, Gaithersburg, MD, USA). The following sense and antisense primers were used for human TSP-1: 5-catgacaaagatggcaaggg-3 (sense), 5-tgctggactggtagccaaag-3 (antisense).

Luciferase Assay  And DNA Construct

To determine TSP-1 promoter activity, we used the THBS 1-2.8 kb promoter-luciferase reporter plasmid purchased from Addgene (plasmid 12,409). The cells were transfected with Lipofectamine (Invitrogen) in the presence of THBSl promoter luciferase reporter plasmid. After drug treatment, the cells were lysed with passive lysis buffer and luciferase activity was measured using dual luciferase kit (Promega, Madison, Wis., USA). The signal was detected using a luminometer (Infinite M200; TECAN, Mannendorf, Switzerland).

Primary seahorse In neurons Dendrite  Spine ( dendritic  spine) density analysis

For dendritic spine analysis, the inventors used two methods: Immunocytochemistry and paloidine staining in the presence of anti-microtubule-associated protein 2B (anti-MAP2B) and anti-vesicle glutamate transporter 1 antibody. Paloidin staining was performed according to the conventional method (Jung et al., 2012, J. Neurosci. 32, 3081-3087). Briefly, primary hippocampal neurons (DIV 21) were fixed with 4% paraformaldehyde in PBS for 10 min at the time of reverse transcription and then incubated with 0.1% Triton X-100 in PBS for 10 min. After washing, the cells were stained with Alexa Fluor 488-phalloidin fluorescent phallotoxin (Invitrogen) in PBS at 4 ° C for 30 minutes. Images of neurons stained with Paloinden were captured with a Konfocal laser microscope (Olympus Fluoview 300), and the dendritic spines were identified with a small protrusion. Densities of dendritic protrusions were measured to have 3-5 dendrites at 8-10 neurons, and the number of spines per unit length of dendrites was quantified and normalized to the dendritic length of 50 μm. Immunocytochemistry was performed according to the conventional method (Crawford et al., 2012, J. Neurosci. 32, 13100-13110) to analyze the dendritic spine density. After the fixation, the primary antibody was incubated with microtubule-associated protein 2B (MAP2B; 1: 200; BD Transduction Laboratories, San Jose, CA, USA) and vesicle glutamate transporter 1 (vGlut1; 1: 100; Neuromab, USA). Primary antibodies were applied overnight at 4 ° C before PBS washing, incubated with AlexaFluor conjugates (1: 500; Life Technologies, Waltham, Mass., USA) for 1 hour, and covered with glass cover. The Z stack of the optical section was visualized with a cone-shaped laser microscope (FV10i-w, Olympus, Japan). At least 10 cultured neurons from two batches per group were used for quantitative analysis.

In the presence of FM4-64 Former synapse Of presynaptic bouton  Functional mark

FM4-64 staining (Invitrogen) was performed according to the manual. Briefly, primary hippocampal neurons were incubated for 1 min at 4 ° C in the presence of 5 mg / mL FM4-64 (Invitrogen) and 50 mM KCl in Hank's balanced salt solution and washed with HBSS to remove free FM4-64.

3D-high resolution SIM (structured illumination microscopy) analysis

To check the TSP-1 level altered by autophagy, the inventors used a high resolution SIM (Nikon N-SIM) according to the existing method (Kook et al., 2012, J. Neurosci. 32, 8845-8854) . Briefly, fixed cells were taken in each 3D-SIM image by moving the stage in the Z-direction with a 0.150 μm step size. The continuous Z-section was reconstituted with a 3D-SIM image (z-axis; brain slice thickness ~ 5.0 +/- 0.4 mu m) and deconvolution was performed in the presence of the alpha-blending function using NIS-E software (Nikon) ) Were performed. Eclipse Ti-E research inverted microscope with Nikon's legendary CFI Apo TIRF 100 oil objective lens (NA 1.49) at 512x512 pixel resolution with an iXon DU-897 EMCCD camera (Andor Technology). Multicolor fluorescence was obtained using a diode laser (488 nm, 561 nm). The exposure time was 40 ms. The electron microscopic gain 150, conversion gain 1, and image were performed with NIS-E software and then sent to Adobe Photoshop.

Immunocytochemical staining

Immunocytochemical staining was performed according to the conventional method (Son et al., 2012, Neurobiol. Aging 33, 1006.e11-1006.e23). Briefly, immobilization with the mouse anti-TSP-1 (1: 300, Santa Cruz), anti-calnexin (1: 500, abcam), or anti-Translocase of outer mitochondrial membranes 20 kDa (1: 300, SantaCruz) Cells were cultured in PBST (Phosphate-buffered saline with Triton X-100) buffer overnight at 4 ° C. After washing several times, the cells were incubated with the secondary antibody, and then images were obtained with a confocal microscope (FV10i-w, Olympus, Japan).

Data Analysis and Statistics

For WB, the protein levels were normalized to the form of a < RTI ID = 0.0 &gt; downskipping &lt; / RTI &gt; protein or pan, such as beta-actin and glyceridehyde 3-phosphate dehydrogenase. All data are expressed as mean standard error. Student's t test was used to compare the 2-groups. After Fisher's LSD post hoc test, analysis of variance was performed using SigmaStat for Windows Version 3.10 (Systat Software, Inc., Point Richmond, CA) And three or more groups were compared. A p value of <0.05 was considered statistically significant.

Example  1. In the hippocampus of the human Alzheimer's brain and Alzheimer's mouse model TSP -1

It is known that TSP-immunoreactive neurons are reduced in the posterior brain of human Alzheimer patients (Buee et al., 1992, Am. J. Pathol. 141, 783e788); However, it is not known which TSP isoforms are decreased and how TSP reduction plays a role in AD pathogenesis. To determine if TSP-1 is reduced in the AD brain, the inventors analyzed TSP-1 levels in brain and AD mouse models of AD patients. In the WB assay, TSP-1 levels at the cortical site of patients with AD were significantly reduced compared to the control (FIGS. 1A and 1B). To confirm TSP-1 level changes in the AD brain, the present inventors used a PS1 double gene mouse that co-expresses Tg6799 mice, APP and / or 5 FAD mutations in an experimental procedure of the conventional method (Oakley et al., 2006, J Neurosci., 26, 10129-10140). Tg6799 mice develop amyloid deposits at 2 months of age and memory impairment at 6 months of age (Oakley et al., 2006, J. Neurosci. 26, 10129-10140).

At 6 and 9 months of age, Tg6799 mice showed decreased levels of TSP-1 in hippocampus compared to nontransgenic littermate controls (Fig. 1C, suppl. 1A). Unlike TSP-1, TSP-2 levels in the Tg6799 brain were unchanged (Fig. 1C). Synapse pathology is an important feature of AD and is associated with memory impairment (DeKosky and Scheff, 1990, Ann. Neurol., 27, 457e464, Honer, 2003, Neurobiol., Aging 24, 1047e1062). Several synaptic proteins have been reported to alter in the AD brain (Jung et al., 2012). PSD-95 and SNP levels in the hippocampus of Tg6799 mice were determined by Western blot to determine if synaptic proteins were altered in the Tg6799 brain. Compared with non-transgenic littermate controls, levels of PSD-95 in Tg6799 mice decreased (Figure 1C; p = 0.0325 for PSD-95). Although not significant, the Tg6799 mice showed a lightweight decrease in SNP levels (Fig. 1C; p = 0.0671 for SNP). PS1 mutations that cause early onset AD cause changes in protein expression levels by impairing lysosomal proteolysis (Ling et al., 2009, PLoS One 4, e4201). To investigate whether the decrease in TSP-1 levels in AD brain is mediated by A? (Amyloid beta), the present inventors used 12-month old Swedish APP-bearing mice (Tg2576) (Hsiao et al., 1996, Science 274, 99e102. ) &Lt; / RTI &gt; brain in the brain. The TSP-1 level in the Tg2576 mouse brain was significantly reduced (Fig. 1D), as compared to the non-transgenic littermate control (Hsiao et al., 1996, Science 274, 99e102), indicating that overexpression of A [beta] is sufficient to cause a decrease in TSP-1 levels. These data demonstrate that levels of TSP-1, as well as synaptic proteins, were reduced in brain and AD pathology-bearing model brain of AD patients.

Implementation 2. The major toxin of Alzheimer's disease A? 1 ~ 42 and TSP -1 relatedness

In the invivo data (Fig. 1) of Example 1, it was shown that TSP-1 levels were reduced in A [beta] pathology. Thus, the present inventors analyzed whether A [beta], one of the major cognates of AD, directly reduced the level of TSP-1. To determine whether Aβ treatment affected the release of TSP-1 in astrocytes, primary astrocytes and U373MG human astroglioma cells were treated with various concentrations of Aβ. Using TCA settling and ELISA, the inventors have found that the level of TSP-1 released into the cell culture medium is reduced by A [beta] (Figures 2A-C) and time (Figure 2D) (Rev-Ap or A? 42-1; Figs. 2A-C). When the level of intracellular TSP-1 was checked, the level of TSP-1 decreased after 24 hours of A? Treatment (Fig. 2D). However, long-term Aβ treatment (72 hours) increased intracellular TSP-1 levels (FIG. 2D). To determine if the levels of other TSPs also change under A [beta] -treated conditions, the present inventors performed Western blot analysis with TSP-2-specific antibodies. Unlike TSP-1, TSP-2 was not altered by Aβ (FIG. 2E), consistent with our Tg6799 brain tissue experiment (FIG. 1C). Next, the present inventors investigated whether there is a clear effect on TSP-1 secretion from other A [beta] astrocytes. It has been reported that oligomeric and fibrotic Aβ plays a different role in astrocytes (White et al., 2005, White, JA, Manelli, AM, Holmberg, KH, Van Eldik, LJ, Ladu, MJ, 2005). Differential effects of oligomeric and fibrillar amyloid-beta 1-42 on astrocytemediated inflammation. Neurobiol. Dis 18, 459e465). The present inventors have found that both A [beta] oligomers and fibers can regulate TSP-1 secretion levels (Fig. 2F). Interestingly, fibrous A [beta] further inhibited TSP-1 secretion compared to the oligomer A [beta] -treated group (Fig. 2F). These results demonstrate that A [beta] decreases TSP-1 secretion from astrocytic cells.

Example  3. Aβ - Induced TSP -1 reduction mechanism

To investigate the mechanism of TSP-1 reduction by A [beta] in astrocytes, we measured the change in TSP-1 transcription levels in the A [beta] -treated state. After carrying out the promoter reporter assay and the reverse transcription polymerase chain reaction, the present inventors found that Aβ plays no role in the transcription of TSP-1 (FIGS. 3A and B); Thus, the present inventors have investigated whether TSP-1 is degraded in cell degradation involving proteasome and / or autophagy-lysosomal pathways. When astrocytes were treated with A [beta], autophagosomes accumulated in the cells (Fig. 3C and D). To determine if Aβ-induced decreases in TSP-1 levels were mediated by autophagy activation, prepared TSP-1 levels were measured after co-treatment of Aβ and autophagy modulating drugs. TSP-1 secretion reduction was alleviated when 3-methyladenine, an autophagy inhibitor, was co-treated with Aβ (FIGS. 3E and F). Furthermore, the activation of autophagy by rapamycin, a well-known autophage-inducing agent, further reduced the amount of TSP-1 secreted into the medium (Fig. 3E) Can be increased. In order to confirm whether the amount of TSP-1 released to the medium by the autophage inhibition alleviates the A? -Mediated reduction, the autophage is knocked down through the knockdown of the autophage-initiating complex, . Compared with the A [beta] -treatment control, the decrease in TSP-1 levels in the media was mitigated by a decrease in beclinc 1 level (Fig. 3F; lane 2 vs. lane 6). To investigate whether A [beta] -induced TSP-1 reduction is mediated by degradation through the proteasome system, MG132, a well-known proteasome inhibitor, was co-applied with A [beta]. Surprisingly, the inventors have found that co-application of A [beta] with MG132 further reduces secreted TSP-1 levels and increases autophagosome formation. Previous studies have shown that inhibition of proteasome activity leads to activation of the autophagy-lysosomal pathway, which implies that there is a complementary relationship between the two systems. For this reason, inhibition of proteasome may induce a decrease in TSP-1 secretion by autophagy activation. Previous studies have shown that metalloproteases such as disintegrin and metalloprotease domain-containing protein 10 (ADAM10) exacerbate at least a portion of TSP-1 degradation extracellularly to regulate TSP-1 levels . We examined whether Aβ-induced reduction of TSP-1 is mediated by proteolysis of TSP-1. 2 broad-spectrum matrix metalloproteinase (MMP) and / or disintegrin and metalloprotease domain inhibitors, TNF-alpha protease inhibitor (TAPI) and GM6001 were applied to astrocytic cultures, 1 is not altered by MMP and / or disintegrin and metalloprotease domain inhibitors (Fig. 3E and F). Immunocytochemistry was performed with TSP-1 using an autopagosomal marker (LC3), an endoplasmic reticulum marker (calnexin), and a mitochondrial marker (20 kDa trans-lokase of the outer mitochondrial membrane) to confirm that TSP- -1. &Lt; / RTI &gt; Using a confocal microscope and SIM, the inventors found that TSP-1 is surrounded by LC3 (FIG. 3G) and is not co-localized with other cell organelles (supp. 2B and C). These data indicate that the mechanism of the level reduction of TSP-1 by A [beta] induction is caused by autophagy activation.

Example  4. TSP Primary seahorse by -1 In a neuron To Aβ  due to Mediated Dendrite  Spine loss prevention

The present inventors have studied the role of TSP-1 in A? -Induced synaptic dysfunction. To study the phenomenon that endogenous TSP-1 released from astrocytes regulates synaptic protein expression in neurons, TSP-1 protein in medium (conditioned media of astrocytes-conditioned medium [ACM]) isolated from cultured astrocytes (3 mg for 6 hours) and / or normal mouse IgG (3 mg for 6 hours; a control for anti-TSP-1 IgG) to neutralize the anti-TSP-1 antibody. After IgG treatment, TSP-1 levels in ACM depleted TSP-1 were found to be reduced by 80% (Fig. 4A). The primary hippocampal neurons cultured for 5 days were supplemented with the solutes (IgG-depleted and TSP-1-depleted medium) and then PSD-95 expression levels were measured by western blotting with or without Aβ . Consistent with previous studies that ACM induces synaptogenesis, the present inventors have found that the addition of ACM to primary hippocampal neurons increases PSD-95 expression (Fig. 4B; lane 1 vs. lane 2 or 4). Treatment of neurons with ACM containing Aβ further reduced PSD-95 levels in the neurons cultured in TSP-1-depleted ACM compared to neurons in normal IgG-depleted ACM (FIG. Lane 5). These results indicate that endogenous TSP-1 prevents A [beta] -induced synaptic dysfunction. Next, in order to determine whether a decrease in TSP-1 release in astrocytes really causes synaptic protein reduction, the present inventors investigated the additional effect of exogenous TSP-1 on synaptic protein expression in the presence of Ap. When the primary hippocampal neurons (DIV 19) were treated with Ap for 48 hours, PSD-95 and NR2A expression levels were significantly reduced (FIG. 4C). Interestingly, co-treatment of recombinant TSP-1 protein and A [beta] alleviated A [beta] -induced decrease in synaptic protein concentration (Fig. 4C). Using mature hippocampal neurons, the inventors further investigated the role of TSP-1 in regulating the dendritic spine density. The neurons were co-labeled with anti-vGluT1 (to stain dendritic spines) and anti-MAP2B (to stain microtubules) antibodies, and vGluT1-positive signals when cells were treated with Aβ (to DIV 19) (Fig. 4D). This means that A [beta] induced a decrease in dendritic spine density. However, co-treatment with TSP-1 protein prevents damage to dendritic spine density (Figure 4D). Paloidin staining was performed to confirm this data. By staining with Paloidin, which is known to bind F-actin in the hippocampal neurons to the thesis (Jung et al., 2012, J. Neurosci. 32,3081e3087), we also found that TSP-1 The role was confirmed. These data demonstrate that dendritic spine injury by Aβ prevents the detrimental effects of Aβ-induction upon treatment with TSP-1.

Example  5. TSP -1 To Aβ  Reduction of functional synaptic activity induced by &lt; RTI ID = 0.0 &gt;

Since TSP-1 restores the A [beta] -induced dendritic spine loss, we have used FM4-64 dye, an amphiphilic fluorescent styryl pyridinium dye, which is used to illuminate the activity-dependent synaptic vehicle recycle in Invitro , Confirming the role of TSP-1 in regulating synaptic activity. Co-culture of brain tough TSP-1 in the presence of A? Relieves the decrease in synaptic activity in A? -Treated cells (FIG. 4E), which suggests that treatment with TSP-1 has a pathological effect of A? -Induced synaptic induction in Invitro It is said to be prevented.

Example  6. TSP -1 In Invivo To Aβ  Of synaptic protein expression changes

To determine if TSP-1 restored A [beta] -induced synaptic dysfunction in invivo, 6-month-old Tg6799 mice and littermates of the same age were injected intraperitoneally into the hippocampus with recombinant human TSP-1 protein &Lt; / RTI &gt; It has been reported that major synaptic dysfunction and A [beta] toxicity are present in the hippocampal subiculum of Tg6799, suggesting that synaptic protein and CREB in the ipsilateral and contralateral hippocampal migration regions of the recombinant TSP- Signaling changes were investigated. TSP-1 ELISA was performed to measure the remaining TSP-1 protein in the brain. The injected TSP-1 protein did not remain in the hippocampal transition 2 weeks after injection (supp 4A), indicating that the elimination process was normal. Two weeks after injection, we found that phosphorylated CREB, total CREB, PSD95, and NR2A levels were significantly increased but NR1 or SNP levels were not (Figures 5A and B). These data indicate that TSP-1 mitigates A [beta] -induced reduction of synaptic proteins such as PSD-95 and NR2A in the in vivo. In investigating whether TSP-1 regulates A [beta] level, the present inventors have found that the A [beta] level in the ipsilateral hippocampal transition is not different from the A [beta] level in the contralateral hippocampal transition (Fig. 5A).

Example  7. To Aβ  Lt; RTI ID = 0.0 &gt; synaptic &lt; / RTI & On a neuron  there is TSP -1 receptor.

The present inventors investigated the role of TSP-1 receptors in A [beta] toxicity in synapses. Several receptors, such as CD47, NL1 and N2l, are known to be receptors of TSP-1 (Graf et al., 2004; Ohnishi et al., 2005, Eroglu et al., 2009). To investigate receptors responsible for TSP-1-mediated protection against A [beta] toxicity, we measured changes in synaptic protein PSD95 levels by KD for candidate receptors. After transfection with cultured hippocampal neurons (DIV9) for 12 days with CACNA2D1 (calcium channel, voltage-dependent, alpha 2 / delta subunit 1) -specific short hairpin RNA (DIV 21) 50% to 60%, and there was no change in β-actin expression. In the? 2? 1 KD neurons, TSP-1 did not mitigate that PSD-95 expression was reduced to A? -induced (Fig. 6A). These results were confirmed by paloidin staining (Figs. 6B and C). In Fig. 6B, A [beta] -induced synaptic loss was mitigated by co-treatment of TSP-I. However, TSP-1 did not restore A? -Induced synaptic loss in the? 2δ1 KD neurons (FIG. 6B, bottom panel), indicating that? 2? 1 is a receptor for TSP-1 under the A? -Treated state. Unlike α2δ1 KD cells, NL1 or CD47 KD cells after combined treatment with Aβ and TSP-1 showed stable PSD-95 expression levels, whereas NL1 and CD47 levels were significantly reduced, but decreased in Aβ-treated cells Respectively. These data demonstrate that TSP-1 mitigates A? -Induced mitigation of synaptic protein through? 2? 1.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (8)

A pharmaceutical composition for preventing or treating Alzheimer's disease comprising TSP-1 (Thrombospondin-1) protein or a gene thereof as an active ingredient.
The method according to claim 1,
Wherein the TSP-1 protein or gene prevents the loss of the dendritic spine caused by amyloid beta in a pharmaceutical composition for preventing or treating Alzheimer's disease.
The method according to claim 1,
Wherein said TSP-1 protein or gene alleviates or ameliorates synaptic protein expression change or activity decrease by amyloid beta.
(Calcium channel, voltage-dependent, alpha 2 / delta subunit 1) modulation kit comprising a TSP-1 (Thrombospondin-1) protein or a gene thereof.
The CACNA2D1 regulatory kit according to claim 4, wherein the nerve cell is a cell in which the dendritic cells are damaged by amyloid beta.
A kit for alleviating or ameliorating a change in synaptic protein expression or activity by amyloid beta in a neuron comprising a TSP-1 (Thrombospondin-1) protein or a gene thereof.
The present invention relates to a method for the treatment of TSP-1 (Thrombospondin-1) protein or a gene thereof in a nerve cell damaged by amyloid beta, subunit 1) modulation method.
A decrease in synaptic protein expression or activity decrease by amyloid beta of neurons in Invitro, comprising the step of treating TSP-1 (Thrombospondin-1) protein or a gene thereof with a dendritic-damaged nerve cell by amyloid beta How to recover.

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