KR20180112649A - Pharmaceutical composition for neurodegenerative brain disease and screening method for the same - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating neurodegenerative brain diseases caused by traumatic brain injuries, which contains a modulator for increasing the expression or activity of calcineurin. The present invention also provides a screening method of a treatment agent for traumatic brain injuries and neurodegenerative brain diseases mediated by tau protein.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative brain diseases and a screening method for the same,

The present invention relates to a neurodegenerative brain disease, more particularly, a kit or a composition for the diagnosis of neurodegenerative brain diseases caused by traumatic brain injury, a pharmaceutical composition for preventing or treating the disease, and screening of the therapeutic agent.

Neurodegenerative brain diseases are degenerative changes in neurons of the central nervous system and cause various symptoms such as impaired motor and sensory function, memory, learning, inhibition of high-order causative functions such as computational reasoning. Representative diseases include Parkinson ' s disease, Alzheimer ' s disease, and memory disorders. Neurodegenerative brain disease is characterized by the rapid or slow progression of necrosis or apoptosis. Therefore, the understanding of the mechanism of death of neurons must be made in order to develop the prevention, control and treatment of central nervous system diseases.

The Tau protein consists of four parts: the N-terminal protruding portion, the proline aggregation domain, the microtubule binding domain and the C-terminal (Mandelkow et al., Acta. Neuropathol., 103, 26-35 , 1996), it is known that when tau is abnormally hyperphosphorylated or transformed in the nervous system of the central nervous system, it induces neurodegenerative brain diseases such as tauopathy.

However, among the conventional neurodegenerative diseases therapeutic agents for treating brain diseases targeting tau protein are not developed much, and the therapeutic agents developed so far mainly target kinase proteins. The precise mechanism by which tau mediated neurodegenerative brain disease is induced is also unknown.

The present invention relates to a therapeutic agent for neurodegenerative brain diseases, in particular neurodegenerative brain diseases caused by traumatic brain injury, more specifically neurodegenerative brain diseases caused by traumatic brain injury mediated by tau protein, The aim of the present invention is to provide a novel target for the treatment of neurodegenerative brain diseases by identifying a specific signal transduction pathway mediating aggregation and neurotoxicity.

The present invention further relates to a kit or composition for the diagnosis of neurodegenerative brain diseases, more specifically neurodegenerative brain diseases caused by traumatic brain injury, by using the signal transduction mechanism of neurodegenerative brain diseases due to such tau mediated traumatic brain injury, Pharmaceutical compositions for preventing or treating such diseases, and screening of said therapeutic agents.

As shown in the following examples, in order to examine how transcriptome changes affect neuropathologic changes of neurodegenerative brain diseases caused by traumatic brain injury, the present inventors used in vitro cell line models and in vivo mice Experiments were performed on the model. Because neurodegenerative brain diseases and Alzheimer's disease (AD) due to traumatic brain injury show similar pathological aspects in terms of increased tau hyperphosphorylation and tauopathy, the present inventors have found that the altered transcriptome signature is associated with traumatic brain injury We analyzed the relationship between neurodegenerative brain disease caused by brain injury and tau phosphorylation pathway in AD. Furthermore, immunoreactivity of p-Tau was confirmed in neurodegenerative brain diseases caused by traumatic brain injury and in post-brain tissue of AD.

"PP2B" is also known as calmodulin-dependent kinase, protein kinase 2B, or PP2B, which is a serine-threonine kinase. Two other subunits A and B bind 1: 1 to form the complete enzyme, which is regulated by Ca ²⁺ / calmodulin.

It is weak against okadadic acid inhibition and the IC ₅₀ is about 10 ^-8 M. The catalytic subunit A has a molecular weight of about 60,000 and a calmodulin binding domain at the C-terminal side. The existence of five species of α1, α2, β1, β2 and β3 is known. And about 50% for all other phosphorylated enzyme molecular species PP1 or PP2A. The regulatory subunit B has a molecular weight of 1.9 million and binds Ca ²⁺ . B has 35% and 29% homology with calmodulin and troponin C, respectively, with amino acids. Cyclosporin A or FK506, which is an immunosuppressant, binds to cyclophilin or FKBP, respectively, and specifically inhibits this enzyme activity.

"Tau" is a microtubule-associated protein, a kind of inrisically disordered proteins (IDPs) that are naturally unstructured. They interact with tubulin to stabilize and promote microtubules do. It is known that when tau protein is overly phosphorylated, NFT (nerofibrillary tangles) is formed and various transconducts are formed, and degenerative brain disease is caused by abnormal aggregation (Lee et al ., Annu . Rev. Neurosci ., 24, 1121-1159, 2001; Bergeron et al, J. Neuropathol Exp Neurol, 56, 726-734, 1997;........ Bugiani et al, J. Neuropathol Exp Neurol, 58, 667- 677, 1999, Delacourte et al ., Ann. Neurol ., 43, 193-204, 1998, Ittner and Gotz, Nat. Rev. Neurosci., 12, 65-72, 2011).

In addition, the tau protein isolated from the microtubules forms a misfoled structure that is easy to bind to other tau proteins. They have undergone aggregation into oligomers, pre-fibrils, which form a β-sheet structure and are bound by several proteins (YS Kim, DJ Kim, O. Hwang, Y. Kim, InTech, 2012, 99-138). Aggregated tau ultimately forms nerve fibers (NFTs) and is called tauopathy throughout the neurodegenerative brain diseases that exhibit these pathological features (DR Williams, Intern Med J 2006, 36 (10), 652 -60). Therefore, dysfunctions due to deformation and aggregation of tau protein are becoming important causes of Alzheimer 's disease and degenerative brain diseases. Agglutination and hyperphosphorylation of abnormal tau directly induces degeneration of neurons, and it is a common pathogenesis of various degenerative brain diseases as well as Alzheimer's dementia.

CTE is a progressive neurodegenerative disease with clinical symptoms including short-term memory loss, Parkinson's, gait, and speech disorders. Various evidence indicates that single or repeated head injury is a risk factor for AD. Importantly, CTE represents a number of neuropathological features, such as tau neuropil neuritis (NNs) and diffuse senile spots. Indeed, the level of abnormally hyperphosphorylated tau protein in neurons and astrocyte of the CTE is similar or identical to the neurofibrillary tangles (NFT) in AD. However, CTE is neuropathologically similar to AD, but how brain damage in CTE causes sequelae of neuropathy is not yet known.

In an example of the present invention, changes in CTE-related transcripts were analyzed. The pathophysiology of CTE involves extensive changes in complex gene expression, but a reduction in PPP3CA expression is observed, particularly in CTE. PPP3CA is a subunit of calcineurin (PP2B) that dephosphorylates tau, and its impairment is related to the formation of amyloid precursor protein and hyperphosphorylated tau, two hallmarks of AD pathology . Previous studies have shown that dysfunction of excitotoxin-induced serine / threonine phosphatase protein activity induces phosphorylation of tau in human neurons and that reduction of calcineurin (PPP3CA) in AD model patients results in tau hyperphosphorylation Mouse model of Huntington's disease in the brain.

Importantly, the experimental system showed that if PPP3CA expression is decreased, tau hyperphosphorylation is likely to occur in CTE, similar to AD. Hyperphosphorylated tau is located in neurofibrillary tangles and axonal arborizations, and the degree of PPP3CA in the region declines in both CTE and AD cases inversely. In vitro experiments using phosphatase inhibitors in BiFC-Tau cells also demonstrated the importance of serine / threonine phosphatase protein activity in tau phosphorylation.

The animal model of TBI imitates repetitive brain damage in humans and offers several advantages, including: Specifically, 1) it is easy to apply repetitive shock continuously, and 2) it exhibits physiological characteristics and symptoms of the disease. Thus, we established an animal model of TBI and studied how PPP3CA knockdown affects the tau phosphorylation of repeated TBI induction in MAPT (P301L) mice. Consistent with the cell model, PPP3CA knockdown using AAV-shPPP3CA significantly increased the p-tau (S202 / T205) immune response in the dentate gyrus of P301L mice after repeated head injury exposure. Together with this, changes in protein phosphatase expression are likely to contribute to CTE tauopathy, which suggests that modulating the expression and activity of serine / threonine protein phosphatase modulates the progressive tau (Tau) pathology of CTE and other neurodegenerative diseases This may be a potential therapeutic strategy to prevent the disease.

The present inventors used PPP3CA (protein phosphatase 3, catalytic subunit, alpha isozyme), PPP3CB (catalytic subunit, beta isozyme) or PP2B (serine / threonine-protein phosphatase 2B) Of phosphorylated (p) -tau proteins is directly related to increased phosphorylated (p) -tau proteins. This finding provides an important insight into PP-dependent neurodegeneration and may lead to the development of new therapies for neurodegenerative brain diseases caused by traumatic brain injury of the tau mediator.

Accordingly, an example of the present invention is a pharmaceutical composition for the prevention or treatment of neurodegenerative brain diseases comprising an agent capable of promoting the expression or activity of Calcineurin, more specifically PPP3CA or PP2B phosphatase (pp) A screening method for the therapeutic agent, a composition or kit for diagnosing a neurodegenerative brain disease, and a disease diagnosis method using the same.

Examples of the neurodegenerative brain diseases include, but are not limited to, neurodegenerative brain diseases caused by traumatic brain injury, Alzheimer's disease, Parkinson's disease, Huntington's disease, etc. In detail, the diseases according to the present invention are Neurodegenerative brain disease caused by aggregation of tau protein or increased phosphorylation of tau protein.

In the present invention, the diagnosis is a concept including both the onset and progress of the disease, and the prognosis after the treatment. In the present invention, the screening refers to selection of a patient who has a possibility of a neurodegenerative brain disease or a patient who includes a risk factor of a neurodegenerative brain disorder, It means to select high patients.

The present inventors have found that the decrease in the activity of PPP3CA or PP2B in neurodegenerative brain diseases caused by traumatic brain injury such as CTE is associated with tauopathy (see Example 4), as described in the following examples, and PPP3CA or PP2B PPP3CA directly controls tau phosphorylation, and PPP3CA or PP2B regulates the dephosphorylation of tau in the CTE model (see Examples 5 and 6). In addition, Respectively. Thus, it can be seen that, when the expression or activity of PPP3CA or PP2B is promoted, phosphorylation or aggregation of tau protein can be inhibited, and thus it can be used as an effective preventive or therapeutic agent for neurodegenerative brain diseases caused by abnormal tau protein .

Specifically, an example of the present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative brain diseases, which comprises an agent capable of promoting the expression or activity of PPP3CA or PP2B.

The agent capable of promoting PPP3CA or PP2B expression or activity refers to a substance which directly or indirectly acts on PPP3CA or PP2B to improve, induce, stimulate or increase the expression or activity of PPP3CA or PP2B. Such a substance means a substance capable of directly or indirectly binding to a gene, mRNA or protein encoding PPP3CA or PP2B to promote the expression or activity of PPP3CA or PP2B, and may be a single compound such as an organic or inorganic compound, a peptide, a protein , An aptamer, an antibody, a nucleic acid, a vector, a biopolymer compound such as a carbohydrate and a lipid, a complex of a natural product and a plurality of compounds, and the like. Preferably, the substance that promotes the expression or activity of PPP3CA or PP2B is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies and natural products.

The mechanism by which the agent promotes the expression or activity of PPP3CA or PP2B is not particularly limited. For example, a substance may act as a mechanism to increase gene expression, such as transcription, translation, or to convert an inactive form to an active form.

For PPP3CA or PP2B with known nucleic acid and protein sequences, those skilled in the art can prepare or screen compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that act as promoters using techniques in the art.

The expression or activity promoter of PPP3CA or PP2B of the present invention may be provided in the form of a vector capable of expressing PPP3CA or PP2B in a cell for use in gene therapy or the like. Accordingly, the present invention relates to a pharmaceutical composition for the prevention or treatment of neurodegenerative brain diseases comprising, as an active ingredient, a nucleic acid or mRNA encoding PPP3CA or PP2B, preferably a recombinant vector containing the nucleic acid.

The nucleic acid sequence encoding PPP3CA or PP2B can be mutated by substitution, deletion, insertion, or a combination thereof, as long as one or more nucleic acid bases encode a protein having equivalent activity. The sequence of such a nucleic acid molecule may be short or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

The vector of the present invention includes, but is not limited to, liposomes, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Examples of preferred viral vectors in the present invention include, but are not limited to, adenovirus, adeno-associated virus, retrovirus, lentivirus, herpes simplex virus, alpha virus). The recombinant vector of the present invention may contain a nucleic acid encoding PPP3CA or PP2B and a regulatory sequence for transcription or translation thereof. A particularly important regulatory sequence is the regulation of transcription initiation, such as promoters and enhancers. It may also contain regulatory sequences consisting of initiation codon, termination codon, polyadenylation signal, Kozak, signal sequence for enhancer, membrane targeting and secretion, IRES (Internal Ribosome Entry Site), and the like. Such regulatory sequences and nucleic acids encoding PPP3CA or PP2B will be operably linked.

The term " operably linked " as used herein means that the linkage between nucleic acid sequences is functionally related. When any nucleic acid sequence is operably linked, any nucleic acid sequence is located so as to be functionally related to another nucleic acid sequence. In the present invention, it is said that when any transcriptional control sequence affects the transcription of a nucleic acid molecule encoding PPP3CA or PP2B, the transcriptional control sequence is operably linked to the nucleic acid molecule. In the present invention, the treatment includes inhibiting or preventing neurodegenerative brain diseases, or reducing, alleviating, reversing symptoms associated with neurodegenerative brain diseases and inhibiting the progression of neurodegenerative brain diseases.

The pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for preventing or treating neurodegenerative brain diseases, which further comprises an appropriate carrier, excipient or diluent commonly used in the production of a pharmaceutical composition. May include non-naturally occuring carriers. Specifically, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .

Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

The content of the agent capable of promoting the expression or activity of PPP3CA or PP2B contained in the pharmaceutical composition of the present invention is not particularly limited but may be from 0.0001 to 50% by weight, more specifically from 0.01 to 20% by weight, % ≪ / RTI > by weight.

In another aspect, the present invention provides a method for preventing or treating neurodegenerative brain diseases comprising administering the pharmaceutical composition to a subject in a pharmaceutically effective amount.

The term " subject " of the present invention may include, without limitation, mammals, aquaculture fish, and the like, including rats, livestock, and humans who are susceptible or susceptible to degenerative brain diseases.

The administration route of the pharmaceutical composition for preventing or treating neurodegenerative brain diseases of the present invention can be administered through any ordinary route as long as it can reach the target tissues. The pharmaceutical composition of the present invention is not particularly limited, but may be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration and the like ≪ / RTI >

The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction to the disease, the age, body weight, sex, history, or kind of the substance used as the active ingredient. For example, the pharmaceutical composition of the present invention may be administered at about 0.1 ng / kg per adult to about 100 mg / kg, specifically about 1 ng / kg to about 10 mg / kg, The frequency is not particularly limited, but may be administered once a day or divided into several doses. The dose is not intended to limit the scope of the invention in any way.

A further embodiment of the present invention relates to a method of treating neurodegenerative brain diseases, comprising the step of measuring the expression or activation of calcineurin, more specifically PPP3CA or PP2B phosphatase, And a screening method of a therapeutic agent for diseases.

More specifically, when the expression or activity of calcineurin in the sample treated with the candidate substance is higher than the expression or activity of calcineurin in the sample not treated with the candidate substance, the candidate substance is caused by traumatic brain injury It is selected as a treatment for neurodegenerative brain diseases.

The neural tissue-derived cells may be neurons or glial cells, and the term " candidate substance for treating neurodegenerative brain diseases " of the present invention is a substance expected to treat neurodegenerative brain diseases, Any substance that can be expected to improve or ameliorate degenerative brain disease can be used without limitation and can be any therapeutic substance such as a compound, a peptide, a peptide mimetic, a protein, an aptamer, an antibody, a natural product, a gene, .

The step of measuring the expression or activation may be performed by any conventional method of measuring the expression level used in the art without limitation, and examples thereof include Western blot, Co-immunoprecipitation assay, Enzyme Linked Immunosorbent Assay (ELISA), real-time RT-PCR, electrophoresis, immunostaining, and fluorescence activated cell sorter (FACS).

The type of neurodegenerative brain disease is not particularly limited, but it is preferably a neurodegenerative brain disease caused by traumatic brain injury. For example, it may be a neurodegenerative brain disease caused by aggregation of tau protein or increased phosphorylation of tau protein .

Another embodiment of the present invention provides a composition for diagnosing neurodegenerative brain diseases caused by traumatic brain injury comprising an agent capable of measuring the inhibition of calcineurin activity. The calcineurin is PPP3CA or PP2B, and the agent may be an antibody or an aptamer capable of specifically binding to PPP3CA or PP2B.

Another embodiment of the present invention provides a kit for diagnosing neurodegenerative brain diseases caused by traumatic brain injury, which comprises an agent for measuring the expression or activity of calcineurin.

The agent to be measured may be a primer or a probe for a calcineurin gene, or an antibody against a calcineurin protein.

Another embodiment of the present invention provides a method for providing information for diagnosing neurodegenerative brain diseases caused by traumatic brain injury, which comprises measuring the inhibition of PPP3CA or PP2B activity. In the step of measuring the inhibition of PPP3CA or PP2B activity, the above-mentioned composition or diagnostic kit for the neurodegenerative brain disease of the present invention can be used. When the activity of PPP3CA or PP2B is inhibited, Degenerative brain disease is diagnosed as having developed.

For the individuals diagnosed with the degenerative brain disease, neurodegenerative brain diseases caused by traumatic brain injury can be prevented or treated by administering the pharmaceutical composition of the present invention.

The present invention provides a novel target for the treatment of neurodegenerative brain diseases by identifying specific signal transduction pathways mediating phosphorylation, aggregation and neurotoxicity of the tau protein.

FIG. 1A is a graph showing the results of analyzing transcript sequences of a traumatic brain injury sample and four normal regions of a normal sample after extracting RNA from each tissue and analyzing the transcript sequence with a next-generation sequencing analyzer to be. The patterns of expression of normal and brain injury samples are distinctly different.
FIGS. 1B and 1C are diagrams showing genes having the same expression pattern by 12-module analysis by simultaneous gene expression analysis using the expression values of the samples. Expression of the genes in the blue module is down in all brain damage samples. Expression of the genes in the remaining modules is increased in both brain injury samples.
FIG. 1D is an analysis of a biochemical pathway-related database of genes belonging to seven modules that are related to brain damage samples among 12 modules. The blue and cyan modules contain the most genes related to degenerative brain disease. In particular, the genes associated with calcium signaling, Huntington and Alzheimer's are highly related.
FIG. 2A shows an analysis of 3,064 genes belonging to the blue module in a biochemical pathway-related database. The 872 genes with increased expression in traumatic brain injury samples are composed of genes related to the MAPK pathway associated with Alzheimer's disease. The 2,192 genes with reduced expression are mainly associated with degenerative brain disease, including nine genes ( CACNA1F , ATP2A2 , GRIN2A , PPP3CA , PPP3CB , PPP3R1 , CALM1 , CALM2 , CALM3 , and CALML3 ) Among them, there is the calcium-dependent phosphatase gene PPP3CA.
Figure 2b shows the expression levels of PPP3CA , PPP3CB , and PPP3R1 in normal and traumatic brain injury samples. All three genes were significantly reduced in brain injury samples.
FIG. 3A shows the expression of PPP3CA and PPP3CB in 11 normal and 11 traumatic brain injury samples, which were not used for sequencing , by qPCR. This data confirms once again that the expression of the two genes in the brain damage sample is reduced.
FIG. 3B is a photograph showing Western blot analysis of protein expression of PPP3CA and PPP3CB in a normal sample and a traumatic brain injury sample. It can be seen that the two proteins are reduced in the traumatic brain injury samples and that the decrease of these two proteins increases the phosphorylation of the tau protein.
3C is a graph of quantitative analysis of the Western blot data of FIG. 3B with a densitometer. PPP3CA and PPP3CB were reduced in exogenous brain injury samples and the phosphorylation of tau protein (S202 / T205) was significantly increased.
Figures 3d and 3e are tauopathies of brain damage samples from a pathological perspective. Immunohistochemical double staining with 3,3-diaminobenzidine (DAB) in brain tissue of the normal sample and exogenous brain injury and quantitative analysis with densitometry revealed that tau phosphorylation was increased in the brain damaged tissue.
FIG. 4A is a graph showing a decrease in expression of PPP3CA gene in Alzheimer's sample by real-time PCR. FIG. It is known that brain damage is similar to Alzheimer 's disease and that Alzheimer' s are also mediated by tauopathy. This is the result of experiments conducted to confirm whether PPP3CA expression is decreased in Alzheimer 's.
FIGS. 4B and 4C are photographs showing protein expression of PPP3CA and PPP3CB in normal and Alzheimer's samples by Western blotting. FIG. It can be seen that both proteins are reduced in Alzheimer's samples, and the decrease of these two proteins shows that the phosphorylation of tau protein is increased. Western blot data were quantitatively analyzed with a densitometer. PPP3CA and PPP3CB are reduced in Alzheimer's sample and phosphorylation of tau protein (S202 / T205) is remarkably increased.
Figures 4d and 4e illustrate tauopathy of Alzheimer's samples from a pathological perspective. Immunohistochemical double staining with 3,3-diaminobenzidine (DAB) in normal samples and brain tissue of Alzheimer's and quantitative analysis with densitometry showed that tau phosphorylation was increased in Alzheimer's tissue as shown in brain injury.
Figure 4f is a graph showing a decrease in the immunoreactivity of PPP3CA and an increase in phosphorylation of tau by confocal microscopy. It was found that PPP3CA and phosphorylated tau protein were present together in Alzheimer cerebral cortical nerve fiber mass.
Figures 5A-5E are graphs showing that protein phosphatase 2B (PP2B) inhibitors increase tau phosphorylation in two cell lines introduced with the tau-BiFC system. The tau gene was ligated to the N-terminal (VN173) or C-terminal fragment (VC155) of the Venus non-fluorescent protein and the engineered tau was stably expressed in the cell line. And a system in which fluorescence appears when two tau are combined. The system was stably expressed in two cell lines (SH-SY5Y and HEK293) and treated with PPAD3CA inhibitors (Okadaic acid, cyclosporine A & Deltamethrin). Increased accumulation of tau phosphorylation and tau in inhibitor treated cells is evidence that PPP3CA is directly involved in tauopathy.
Figures 6a and 6b show that PPP3CA regulates the dephosphorylation of tau protein in cell models. GFP-PPP3CA, GFP-GSK3B and tau-GFP were transfected into the cells. At this time, GFP-PPP3CA was experimented in a concentration-dependent manner. Proteins were extracted from these cells and subjected to Western blotting. As a result, phosphorylation of tau decreased and GSK3B protein expression amount decreased depending on the treatment concentration of PPP3CA. These results demonstrate that PPP3CA regulates phosphorylation of tau protein.
FIG. 6c is a graph showing that PPP3CA regulates the dephosphorylation of tau protein. The construct was constructed by deleting the catalytic site of PPP3CA, and the protein was extracted and subjected to Western blotting. As a result, there is no change in phosphorylation of tau protein.
FIG. 6d is a graph showing that PPP3CA regulates the dephosphorylation of tau protein. The shRNA construct of PPP3CA was prepared and transfected into cells, followed by Western blotting. As a result, PPP3CA was reduced and tau protein phosphorylation was increased. These results demonstrate that PPP3CA regulates the phosphorylation of tau protein as shown in Figures 6a and 6b.
Figure 6e shows that PPP3CA / PP2B regulates phosphorylation of tau protein in a mouse model system. The shRNAs of PPP3CA were inserted into adeno-associated viral (AAV) vectors and constructed into hippocampal regions of tau (P301L) transgenic mice. I traumatized the mouse head several times in a weight drop manner. Like the cell model, mice with significantly reduced expression of PPP3CA in AAV-shPPP3CA mice were obtained.
FIG. 6f is a graph showing that phosphorylation of tau protein is regulated depending on the concentration of PPP3CA in the mouse obtained in FIG. 6e. FIG. There was an increase in tau phosphorylation in the hippocampal region of traumatized AAV-shPPP3CA mice. As a result, PPP3CA also demonstrated the phosphorylation of tau protein in animal models as well as in cell models.
Figure 6g shows that the shRNA of PPP3CA was inserted into adeno-associated viral (AAV) vectors and then inserted into hippocampal regions of tau (P301L) transgenic mice. I traumatized the mouse head several times in a weight drop manner. As a result, tau phosphorylation was also increased in the dentate gyrus of hippocampus.
Figure 6h is a schematic diagram illustrating the entirety of the present invention. In extrinsic brain damage samples, PPP3CA / PP2B increases phosphorylation of tau protein and increases oligomer formation of phosphorylated tau protein. As a result, neurofilament tangles are formed in brain damaged samples, resulting in loss of neurons.
FIG. 7 is a diagram showing the expression of genes involved in three cell types, ie, neurons, astrocytes, and rare prominent glial cells, in order to examine whether the traumatic brain injury is a cell-specific phenomenon. As can be seen from the results, the gene expression involved in neurons is expressed low in the traumatic brain injury sample whereas the expression of the genes related to the astrocyte and rare prominent glue cells is high.
FIG. 8A is a graph showing analysis of genes belonging to the blue module among co-expressed gene modules revealed by gene network analysis. FIG. The genes belonging to the blue module showed the highest correlation in traumatic brain injury samples.
Figure 8b is a Heatmap showing the expression of genes in the blue module in traumatic brain injury and normal samples. Green means relatively low expression and red means high.
FIG. 8C is a heatmap diagram showing the expression of genes associated with tau phosphorylation in each module.
9 is a western blot figure in which PPP3CB decreases the level of tau phosphorylation in a concentration-dependent manner. As a result, the level of phosphorylated tau is inversely proportional to the concentration of PPP3CB.
Figure 10 is a graph showing that repeated traumatic brain injury increases phosphorylation of tau protein in the hippocampal region of Tau transgenic mice. AAV-shRNA-PPP3CA increased tau phosphorylation in the mouse brain CA1, CA2, CA3 and dentate gyrus (DG) and showed no change in AAV-shRNA.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Experimental methods and materials for the experiment of the present invention will be described first as a reference example.

Reference example  1: Human tissue

Neuropathological processing of human brain samples with controls, Alzheimer's Disease (AD) and Chronic Traumatic Encephalopathy (CTE) was performed following established procedures at Boston University Alzheimer's Disease Center (BUADC) and Chronic Chronic Traumatic Encephalopathy (CTE) Respectively. Approval of the Institutional Review Board for ethical clearance was obtained through the BUADC and CTE centers. The study was reviewed by the Boston University School of Medicine Institutional Review Board (Protocol H-28974) and approved for exemption from studies using only tissues collected from post-mortem subjects not classified as human subjects. Next, relatives gave their participation in brain donation and informed consent. This study was conducted in accordance with the guidelines and principles of institutional regulation of the protection of human subjects in the Helsinki Declaration. Detailed information on brain tissue is provided in Table 1 (clinical information under test) and in Table 2 (age, sex, and Braak stage of AD patients). In all cases diagnosed with autopsy, AD was specified as the cause of death.

Samples individual Location Diagnosis Age / Sex CTE  Stage TDP -43 SF_T1
T1
Superior Frontal


CTE



84 / M


4


4 AT_T1 Anterior Temporal PV_T1 Posterior Visual SP_T1 Superior Parietal SF_T2
T2

Superior Frontal


CTE


73 / M


4


3 AT_T2 Anterior Temporal PV_T2 Posterior Visual SP_T2 Superior Parietal AT_T3
T3
Anterior Temporal

CTE

80 / M

4

One PV_T3 Posterior Visual SP_T3 Superior Parietal SP_T4 T4 Superior Parietal CTE 73 / M 3 One SF_T5 T5
Superior Frontal
CTE
76 / M
4
2 AT_T5 Anterior Temporal SF_T6
T6
Superior Frontal

CTE



93 / M


4


4
AT_T6 Anterior Temporal PV_T6 Posterior Visual SP_T6 Superior Parietal SF_C1
C1
Superior Frontal

Normal

88 / M

irrelevant

irrelevant AT_C1 Anterior Temporal SP_C1 Superior Parietal SF_C2
C2
Superior Frontal


Normal


82 / M


irrelevant


irrelevant AT_C2 Anterior Temporal PV_C2 Posterior Visual SP_C2 Superior Parietal SF_C3
C3

Superior Frontal


Normal


74 / M


irrelevant


irrelevant AT_C3 Anterior Temporal PV_C3 Posterior Visual SP_C3 Superior Parietal SF_C4
C4

Superior Frontal


Normal


67 / M


irrelevant


irrelevant AT_C4 Anterior Temporal PV_C4 Posterior Visual SP_C4 Superior Parietal SF_C5
C5

Superior Frontal


Normal


87 / F


irrelevant


irrelevant AT_C5 Anterior Temporal SP_C5 Superior Parietal PV_C5 Posterior Visual AT_C6 C6 Anterior Temporal Normal 70 / M irrelevant irrelevant SF_C7
C7

Superior Frontal


Normal




63 / M



irrelevant


irrelevant AT_C7 Anterior Temporal SP_C7 Superior Parietal PV_C7 Posterior Visual

Number Case Age Sex Break stage One Normal 87 F Ⅰ 2 Normal 88 M Ⅰ 3 Normal 86 M Ⅱ 4 Normal 87 F Ⅱ 5 Normal 67 M Ⅰ 6 Normal 82 M Ⅰ 7 Normal 70 M Ⅰ 8 Normal 88 M Ⅰ One AD 79 F VI 2 AD 70 M VI 3 AD 80 F V 4 AD 92 M V 5 AD 75 M V 6 AD 83 M VI 7 AD 79 F VI 8 AD 89 M I-V

Reference example  2: RNA sequence analysis

Samples were prepared using the Illumina TruSeq RNA sample preparation kit according to the manufacturer's instructions and sequenced on a HiSeq 2000 platform (Illumina, San Diego, Calif., USA). The 101_bp base sequence pair reading sequence was mapped to the hg19 reference human genome using the STAR 2-pass 15 method. The inventors used HTSeq to calculate the number of readings aligned to each gene based on Ensembl gene set 16.

We excluded samples that failed in library preparation or sequencing and also excluded samples with less than 10 million readings. Overall, 18 patients with CTE and 24 normal subjects were examined. The normalized number of readings was performed through R and Cluster 3.0 and applied to PCA or clustering analysis visualized via Java Treeview (Anders S, Pyl P, Huber W. HTSeq-A Python framework to work with high-throughput sequencing data. Bioinformatics 2015; 31: 166-169; de Hoon MJ, Imoto S, Nolan J, Miyano S. Open source clustering software. Bioinformatics 2004; 20: 1453-1454; Saldanha AJ Java treeview - extensible visualization of microarray data. 2004; 20: 3246-3248)

Reference example  3: Weighted gene analysis coexpression  network analysis)

Co-expression analysis was performed using the weighted gene co-expression network analysis (WGCNA) method with R package (v3.0.0) (Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 2008 ; 9: 559). Overall, 24,682 genes were used to construct each network. To construct the network, the absolute value of the Pearson's correlation coefficient was calculated for all possible gene pairs, and the final value was modified so that the final matrix conformed to the approximate scale-free topology. The connectivity (k) of each gene was calculated by summing the link strength with other genes.

A module is defined as a cluster of closely linked genes. By default, the module is marked as a branch of the systematic clustering tree using the difference measure. Each module is subsequently color-coded. The gene expression profile of each module was summarized according to the module eigengene (defined as the first major component of the module expression level), defined as the first major component of the module expression level. The significance (GS) measure of the gene was calculated to evaluate the correlation with the phenotype. Intra-modular connectivity (k-within) was calculated for each gene by summing the linking strengths of other module genes and dividing them by the maximum intra-module connectivity. The adjacent threshold to include the edge of the printout was set to 0.2. The eigengene-based connectivity of a gene in a module is defined as the correlation between the gene and the corresponding module eigengene.

Reference Example 4: Enrichment analysis based on KEGG

Functional annotation analysis was used to establish a biological relationship with the gene network module using the Gene Set Enrichment Analysis (GSEA) with the KEGG pathway (Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA et al Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 2005; 102: 15545-15550). The enrichment of KEGG term in each module was evaluated based on the high speed test. The outcome of the GSEA is an enrichment score indicating the imbalance of gene expression ranking distribution in each gene set. The number of genes of superposition (k) was set to 3 or more. The enrichment score was normalized according to the size of the gene set and then graded according to the normalized enrichment score. The default False Positive Rate (FDR) q value setting (FDR q value <0.05) was used as a cut-off.

Reference Example 5: Tau BiFC cell line and culture

Establishment of the HEK293 / tau-BiFC cell line has been described in the previously reported document (Tak H, Haque MM, Kim MJ, Lee JH, Baik JH, Kim Y et al Himolecular fluorescence complementation, living cells. PLOS One 2013; 8: e81682). The amino-terminal fragment (VN173) and the carboxyl-terminal fragment (VC155) of Venus fluorescence protein, independently of each other, were fused to the full-length tau protein. For neuronal cell expression, the tau-BiFC constructs (pCMV6-hTau40-VN173 and pCMV6-hTau-VC155) were transfected with SH-SY5Y. SH-SY5Y / tau-BiFC Stable cells were selected using Geneticin (200 μg / ml). Phosphorylation of tau resulted in the aggregation or aggregation of two tau molecules that allowed the maturation of the Venus fluorescence protein to emit a fluorescent signal. All established cell lines were cultured in a humidified atmosphere containing 37.5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10,000 units / ml penicillin, 10,000 占 퐂 / ml streptomycin and 1 占 퐂 / ml Geneticin.

Reference example  6: Live cell imaging and analysis

For microscopic image analysis, HEK293 tau-BiFC and SH-SY5Y tau-BiFC cells were plated on black 384-well plates. The next day, tau-BiFC cells were treated with various concentrations of okadaic acid (O8010, Sigma), cyclosporin A (C3662, Sigma) or deltamethrin (D9315, Sigma). After incubation for 29 hours, the plates were imaged using an Operetta high contents imaging system (PerkinElmer). Cellular intensities of Tau-BiFC fluorescence were analyzed using Harmony 3.1 analysis software. Each experiment was performed three times. The bar graph for BiFC intensity represents the mean ± SEM in three independent experiments.

Reference Example 7: Confocal microscopy

According to the chamber reported previously (Rahman A, Grundke-Iqbal I, Iqbal K. PP2B isolated from human brain preferentially dephosphorylates Ser-262 and Ser-396 of the Alzheimer disease abnormally hyperphosphorylated tau J Neural Transm. 2006; Tau (S202 / T205) (mouse monoclonal, 1: 500, Thermo Scientific) and PP2B / PPP3CA (rabbit polyclonal, , 1: 200, SantaCruz Biotech.) Were immunostained for tissue and cells. For confocal microscopy analysis, specimens were incubated with primary (FITC) -conjugated secondary antibodies (Vector, Burlingame, CA) and Cy3-linked secondary antibodies (Jackson Lab) for 1 hour after primary antibody culture . Images were analyzed using a spinning disk confocal microscope (IX2-DSU, Olympus). Pre-absorption by an excess of target protein or omission of the primary antibody was used to demonstrate antibody specificity and to determine the background generated in the detection assay.

Reference example  8: Immunohistochemical analysis Immunohistochemistry )

Immunohistochemical analysis was performed as previously described (Lee J, Hwang YJ, Shin JY, Lee WC, Wie J, Kim KY et al. Epigenetic regulation of cholinergicreceptor M1 (CHRM1) by histone H3K9me3 impairs Ca 2 + signaling in Huntington ' s disease Acta Neuropathol 2013; 125: 727-739). The paraffin embedded tissues were incised in 10-20 ㎛ coronal plane. Tissue sections were rehydrated and blocked with brothing solution (1% H2O2), then p-Tau (S202 / T205) (1: 200), PP2B / PPP3CA (1: 200, SantaCruz Biotech) 1: 200, Abcam) and an anti-III typing antibody (1: 500 dilution, SIGMA) for 24 hours. After three washes, the slides were treated with a vector ABC kit (Vector Lab). Immunoreactivity signals were measured with a DAB chromogen (Thermo Fisher Scientific, Meridian, Rockford, USA) and analyzed with a brightfield microscope.

Reference Example 9: Western blot analysis [

Western blot analysis was performed as described above (Rahman A, Ting K, Cullen KM, Braidy N, Brew BJ, Guillemin GJ et al., The excitotoxin quinolinic acid induces tau phosphorylation in human neurons. . p-Tau (S199) (1: 1000; AbCam), anti-p-Tau (S396) (1: 1000), anti-p-Tau (Pierce, 170-6515 and 170-6516) conjugated to horseradish peroxidase after treatment with anti-beta-actin (1: 10000; Sigma Aldrich) An enhanced chemiluminescence (ECL) kit (Thermo Scientific) was used to detect the immune response.

Reference example  10: Quantitative real time Polymerase chain reaction (Quantitative real-time PCR , qPCR)

Total RNA was extracted from frozen brain tissue using TRIzol reagent (MRC, TR118) (Lee J, Hwang YJ, Shin JY, Lee WC, Wie J Kim KY et al.) Epigenetic regulation of cholinergicreceptor M1 (CHRM1) by histone H3K9me3 impairs Ca2 + signaling in Huntington's disease Acta Neuropathol 2013; 125: 727-739.). 50 ng of RNA was used as a template for quantitative RT-PCR amplification using SYBR Green Real-Time PCR Master Mix (Toyobo, QPK-201, Japan). Prior to the start of the plateau, the primers were normalized in a linear range of cycles. Primer sequences are shown in Table 3 (information of primers used in qPCR to view gene expression).

Gene direction Oligo Sequence SEQ ID NO: PPP3CA
(catalytic domain) Forward 5'-GATGAAGCTCTTTGAAGTCG-3 ' One Reverse 5'-AGTCAAAGGCATCCATACAG-3 ' 2 PPP3CA
(Exon 13) Forward 5'-CTAGAAGTCCCCAATGCAGTA-3 ' 3 Reverse 5'-TCTTGCCTCTGACAGTAGCTT-3 ' 4 PPP3CA
(inhibitory domain) Forward 5'-AGGAAGAAGCCTGTGGAAAT-3 ' 5 Reverse 5'-GAATTCTCATCCTTCCACTGA-3 ' 6 PPP3CB
Forward 5'-GTGAAAGAAGGTCGAGTAGA-3 ' 7 Reverse 5'-GGTATCGTGTATTAGCAGGT-3 ' 8 PPP1CA
(catalytic domain) Forward 5'-AGAAGACGGCTATGAGTTCT-3 ' 9 Reverse 5'-GTACTTCCCCTTGTTCTTGT-3 ' 10 GAPDH
Forward 5'-GAAATCCCATCACCATCTTCC-3 ' 11 Reverse 5'-GAGGCTGTTGTCATACTTCTC-3 ' 12

GAPDH was used as an internal control. Real-time data acquisition was performed using the LightCyler 96 Real-Time PCR System (Roche Diagnostics, Germany) at cycle conditions of 1 min x 1 cycle at 95 ° C, 15 sec at 95 ° C, 1 min 45 sec cycle at 60 ° C did. LightCyler96 software was used to analyze the relative gene expression and indicate the number of cycles required to generate a fluorescence signal above a predefined threshold in Ct.

Reference example  11: PPP3CA shRNA  Included rAAV  Creation and delivery of vectors

For in vivo gene silencing, the mouse shRNA sequence for PPP3CA was cloned into the pSicoR vector using the HpaI / XhoI site (Addgene # 21907) and the pAAV-MCS vector (Stratagene) using the MluI / BglII site ). High-potency rAAV vectors were produced in HEK293TN cells using a helper virus-free system. Briefly, after transfection with the same molar rep / cap / helper plasmid, the rAAV vector was produced. After 72 hours of incubation, cells were lysed, treated with benzonase (Sigma # E1014) and further purified using HiTrap heparin column (GE healthcare # 17-0460-01). And concentrated to a final volume using an Amicon ultra-15 centrifugal filter device (Millipore # UFC9100). A stereotactic microinjection method was used to deliver rAAV-PPP3CA shRNA to the brain. As previously described, rAAV-shControl and AAV-PPP3CA shRNA were delivered to tooth marrow (AP: -2, ML: 1.5, DV: -1.85) of 4 month old wild-type mice and Tau transgenic (P301L) mice.

Reference Example 12: TBI animal model

All procedures for animal testing have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Science and Technology in accordance with international standards and guidelines. Four-month old wild-type (C57BL / 6) and Tau-transgenic (P301L) mice were used in this study. Rats fed a standard laboratory diet and water in a controlled environment. Mice received three weight-loss induction closure spreading TBI at 3-day intervals. We used a weight reduction device (weight 100 g, drop height 75 cm, angle 90 degrees) as described above to induce closed diffusion TBI (25). The anatomical location was adjusted to bregma -1 to ± 4. All mice were initially anesthetized with IP injection of 2% avertin (23 μg / g) and received weight loss induced TBI. Sham-injured animals received the same protocol of anesthetic administration, but never lost weight. After TBI, the mice were placed in a supine position in a clean, heated cage using a commercially available heating pad. After normal behavior (eg, grooming, walking, exploration) was restored, we returned the mouse to the original us. We investigated animal behavior based on open fields and assessed neuro-histological changes. Animals were euthanized 24 hours after the last shock and sham-operated animals were euthanized 24 hours after the last anesthetic.

Example  One: CTE Transcript  analysis

Previously, the potential contribution of gene expression to the pathogenesis of CTE has never been studied. The present inventors have identified four regions of posterior brain tissue (anterior temporal (AT), posterior visual (PV), and posterior visual (CT) phases from six subjects and seven normal controls with neuropathologically confirmed CTE stages III and IV, ), The superior frontal (SF), and the superior parietal (SP) cortical tissue). RNA sequencing was then performed (see Table 1).

 Paired-end RNA-Seq was performed using the Illumina HiSeq 2000 platform, and the sequencing readings were aligned to the hg19 reference human genome. The average number of readings was 71 million (74 million average for normal subjects and 76 million for CTE samples), and approximately 77.4% of the total readings were mapped to human transcripts (Table 4-1 and Table 4-2 ).

Samples Read Length (bp)  # of Reads Bases Uniquely mapped reads ( % )  # of Uniquely    mapped reads Mapped Bases SF_T1 2 X 101 89,477,966 9,037,274,566 79.41% 71,051,654 7,176,217,054 AT_T1 2 X 101 82,974,818 8,380,456,618 76.06% 63,112,408 6,374,353,208 PV_T1 2 X 101 72,850,874 7,357,938,274 74.44% 54,232,254 5,477,457,654 SP_T1 2 X 101 59,511,698 6,010,681,498 75.32% 44,825,706 4,527,396,306 SF_T2 2 X 101 55,326,844 5,588,011,244 79.39% 43,921,866 4,436,108,466 AT_T2 2 X 101 76,292,538 7,705,546,338 79.89% 60,947,122 6,155,659,322 PV_T2 2 X 101 52,789,162 5,331,705,362 75.92% 40,078,716 4,047,950,316 SP_T2 2 X 101 66,284,570 6,694,741,570 78.17% 51,815,358 5,233,351,158 AT_T3 2 X 101 88,249,746 8,913,224,346 82.20% 72,538,134 7,326,351,534 PV_T3 2 X 101 104,741,092 10,578,850,292 82.97% 86,903,176 8,777,220,776 SP_T3 2 X 101 68,224,762 6,890,700,962 77.06% 52,574,352 5,310,009,552 SP_T4 2 X 101 91,831,500 9,274,981,500 86.16% 79,118,050 7,990,923,050 SF_T5 2 X 101 80,498,582 8,130,356,782 81.93% 65,950,646 6,661,015,246 AT_T5 2 X 101 94,223,304 9,516,553,704 79.44% 74,851,080 7,559,959,080 SF_T6 2 X 101 77,081,788 7,785,260,588 78.87% 60,795,150 6,140,310,150 AT_T6 2 X 101 81,407,012 8,222,108,212 69.02% 56,189,194 5,675,108,594 PV_T6 2 X 101 47,711,984 4,818,910,384 78.47% 37,440,362 3,781,476,562 SP_T6 2 X 101 79,633,890 8,043,022,890 71.99% 57,324,582 5,789,782,782 SF_C1 2 X 101 67,647,636 6,832,411,236 57.26% 38,737,874 3,912,525,274 AT_C1 2 X 101 74,646,302 7,539,276,502 91.36% 68,196,390 6,887,835,390 SP_C1 2 X 101 70,227,800 7,093,007,800 77.35% 54,317,972 5,486,115,172 SF_C2 2 X 101 76,371,856 7,713,557,456 60.67% 46,337,958 4,680,133,758 AT_C2 2 X 101 85,536,650 8,639,201,650 75.36% 64,461,774 6,510,639,174 PV_C2 2 X 101 84,707,524 8,555,459,924 65.46% 55,447,140 5,600,161,140 SP_C2 2 X 101 87,699,934 8,857,693,334 77.70% 68,139,492 6,882,088,692 SF_C3 2 X 101 86,123,180 8,698,441,180 75.58% 65,091,718 6,574,263,518 AT_C3 2 X 101 99,450,720 10,044,522,720 81.71% 81, 264, 282 8,207,692,482 PV_C3 2 X 101 90,908,222 9,181,730,422 70.12% 63,744,808 6,438,225,608 SP_C3 2 X 101 112,046,048 11,316,650,848 62.38% 69,894,916 7,059,386,516 SF_C4 2 X 101 69,153,502 6,984,503,702 81.46% 56,334,952 5,689,830,152 AT_C4 2 X 101 86,533,640 8,739,897,640 79.55% 68,837,332 6,952,570,532 PV_C4 2 X 101 64,090,504 6,473,140,904 76.49% 49,020,506 4,951,071,106 SP_C4 2 X 101 49,689,838 5,018,673,638 57.41% 28,525,606 2,881,086,206 SF_C5 2 X 101 53,770,430 5,430,813,430 87.23% 46,905,748 4,737,480,548 AT_C5 2 X 101 61,453,168 6,206,769,968 88.65% 54,475,924 5,502,068,324 SP_C5 2 X 101 67,454,288 6,812,883,088 78.49% 52,947,456 5,347,693,056 PV_C5 2 X 101 81,778,604 8,259,639,004 88.67% 72,509,850 7,323,494,850 AT_C6 2 X 101 62,723,588 6,335,082,388 83.23% 52,201,812 5,272,383,012 SF_C7 2 X 101 74,618,218 7,536,440,018 80.35% 59,953,846 6,055,338,446 AT_C7 2 X 101 43,290,416 4,372,332,016 86.18% 37,309,556 3,768,265,156 SP_C7 2 X 101 62,895,188 6,352,413,988 90.56% 56,955,946 5,752,550,546 PV_C7 2 X 101 67,292,522 6,796,544,722 90.14% 60, 654, 426 6,126,097,026 AT_AD1 2 X 101 73,765,170 7,450,282,170 78.71% 58,061,442 5,864,205,642 AT_AD2 2 X 101 83,992,030 8,483,195,030 81.82% 68,722,096 6,940,931,696 AT_AD3 2 X 101 51,885,624 5,240,448,024 79.58% 41,288,224 4,170,110,624 AT_AD4 2 X 101 46,649,484 4,711,597,884 65.74% 30,667,548 3,097,422,348

Principal component analysis (PCA) was performed on the RNA-seq samples to evaluate the similarities and differences between the samples. As a result, it was confirmed that the transcript pattern (FIG. 1A) between the CTE and the normal group was clearly distinguished.

Example 2: Identification of gene modules co-expressed by gene network analysis

The present inventors performed weighted gene co-expression network analysis (WGCNA) 19 to identify CTE-related co-expression modules and key components. WGCNA allows biologic interpretation of transcription patterns by assembling genes with similar expression patterns in a biased manner. The present inventors identified twelve distinct co-expression modules with different gene sizes from 74 to 3,355. A total of 13,331 genes in the network did not share similar co-expression with other genes and did not belong to any module (Fig. 1b).

module eigengene represents the expression of all genes classified into the module. Using Pearson's method, each eigengene expression was correlated with experimental variables such as CTE status or brain regions (AT, PV, SF and SP). Only CTE status was positively or negatively correlated with module eigengenes (Fig. 1C). Blue modules (r = -0.87, P = 8x10-14) were negatively correlated with CTE subjects, and many of the genes included in this module were down-regulated in the CTE brain. On the other hand, other important modules of the CTE showed a positive correlation. As a result of changes in the expression patterns of cell type-specific genes, we confirmed that genes related to neurons and oligodendrocytes were affected by CTE progression. Specifically, the neuron-related gene was contained in a large amount in the blue module, and the expression was decreased in the CTE (FIG. 7). This was consistent with the PCA analysis, and WGCNA analysis showed no significant difference in the four brain regions. Only AT and PV showed positive correlation with purple and black modules, respectively.

The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Figure 1d) and gene set enrichment analysis (GSEA) were performed to understand the biological relevance of the highly correlated modules with CTE. Two of the seven modules (blue and cyan modules) were highly enhanced in certain functional pathways that are directly related to neurodegenerative diseases and neurophysiological functions. In particular, the blue module contains many genes belonging to the calcium-signal pathway, Alzheimer's disease, Huntington's disease, Parkinson's disease and the MAPK signaling pathway. The brown and black modules were rich in genes belonging to pathways involved in cancer, focal adhesion, and actin cytoskeleton regulation. This analysis confirmed that a large regulatory change in gene expression was associated with the CTE brain.

Example  3: blue  The gene co-expression module CTE and  Significantly related

The blue module contains 3,064 genes with high correlation between module members and gene importance for CTEs (r = 0.85 and P < 1x10-200, Figure 8a). Unsupervised hierarchical clustering performed at the gene expression level of the blue module revealed a clear separation of normal individuals and CTE (Fig. 8b). In CTE, 872 upregulated genes and 2,192 downregulated genes were abnormally expressed. Functional annotation analysis based on the KEGG pathway using the upregulated and downregulated genes in the blue module is shown in Figure 2a. Upregulated genes are mainly involved in the MAPK pathway associated with the pathogenesis of Alzheimer's disease. However, tau phosphorylation kinase, p38 MAPK (MAPK11, MAPK12, MAPK13 and MAPK14) were not activated or did not bind to the blue module. In addition, the expression of the other tau phosphorylation kinases GSK3 (GSK3A and GSK3B), CDK5 and FYN did not increase in the CTE (Fig. 8C). Downregulated genes are associated with various neurodegenerative diseases. Among these genes, we focused on genes that are not related to Huntington's disease and Parkinson's disease that do not display tauopathy for major disease progression. Using this approach, we identified nine genes that could affect CTE progression: CACNA1F, ATP2A2, GRIN2A, PPP3CA, PPP3CB, PPP3R1, CALM1, CALM2, CALM3 and CALML3. The present inventors confirmed PPP3CA encoding calcium-dependent, calmodulin-stimulated protein phosphatase from the above-mentioned genes, and it was reported that the decrease in the temporal lobe of the brain of AD patients There is a bar. In addition, PPP3CB and PPP3R1, subunits of PP2B, were significantly reduced in CTE subjects (Fig. 2B).

Example  4: PPP3CA  / PP2B  The decrease in activity CTE Tau  It is related to the pathology.

The present inventors have examined whether changes in PPP3CA gene expression contribute to pathological tau phosphorylation and tauopathy of CTE. First, decreased expression of PPP3CA was confirmed by qPCR in human CTE and cortex of normal sample (Fig. 3a). Western blot and densitometry analysis showed that the concentration of PP2B protein was significantly reduced in CTE compared to normal (FIGS. 3b and 3c). In addition, the levels of p-tau in S199, S202 / T205, and S396, the most common sites of tau phosphorylation in neurodegenerative diseases, increased 3-30 fold in CTE. Importantly, the levels of PP2B protein and p-tau (S202 / T205) in the prefrontal cortex of patients with CTE are inversely related. To further study the pathological features of tauopathy in CTE, we performed 3,3'-diaminobenzidine (DAB) immunohistochemistry in the cortex of normal and CTE brains (FIGS. 3d and 3e). As a result, it was confirmed that the immunoreactivity of PPP3CA was decreased and the tau was hyperphosphorylated in CTE (S199, S396, S205 / T208) in accordance with qPCR and Western blot data.

Example  5: PPP3CA  / PP2B  The decrease in activity was Tau  It is related to the pathology.

Because the CTE features are clinically and pathologically closely overlapped with AD, the present inventors have investigated whether there is a possibility that PP2B activity is also decreased in ADs exhibiting the most common tau pathologies.

We performed qPCR on PPP3CA in AD subjects and confirmed that expression was reduced as shown in the CTE subjects (Figure 4a). We observed a similar inverse correlation between PPP3CA levels and p-tau in AD as observed in CTE (Fig. 4b and c). As expected by the present inventors, not only the immunoreactivity of PPP3CA in AD was reduced, but also the tau protein was hyperphosphorylated (S199, S396, S205 / T208) (Fig. 4d and 4e). Confocal microscopy showed a decrease in the immunoreactivity of PPP3CA, while the immunoreactivity of p-tau was increased in AD (Fig. 4f). In addition, the present inventors confirmed that p-tau (S202 / T205) and PPP3CA coexist in the cortex of AD.

Example  6: PPP3CA  / PP2B  Active inhibition Tau  Phosphorylation and Tau  Induced aggregation.

After confirming reduced PPP3CA activity and elevated tau phosphorylation in human CTE and AD samples, the present inventors used a tau pathology in vitro cell line model to investigate whether the PPP3CA inhibitor is sufficient to increase phosphorylation and aggregation of tau. To this end, the present inventors observed tau phosphorylation and aggregation with fluorescent images using Tau-BiFC cell lines (SH-SY5Y and HEK293) (Fig. 5A). Three different PP2A / PPP3CA inhibitors (okadaic acid, cyclosporine A, and deltamethrin) increased tau phosphorylation levels in both SH-SY5Y and HEK293 Tau-BiFC cell lines (Fig. Treatment with okadaic acid and cyclosporine A increased tau phosphorylation level in proportion to concentration, while deltamethrin tended to decrease after a threshold of 3 μM (Fig. 5c). Similar results were observed in the HEK 293 cell line and the tau phosphorylation intensity due to okadaic acid was higher (Figure 5d).

Example  7: PPP3CA Tau  Directly regulates phosphorylation.

After confirming the relevance of PPP3CA catalytic activity to tau dephosphorylation, the present inventors confirmed whether PPP3CA directly controls tau phosphorylation (FIG. 6). Since GSK3 is a well-known tau kinase, we overexpressed GSK3 with PPP3CA and tau and determined the level of phosphorylated tau. While overexpression of GSK3 increased tau phosphorylation in S202 / T205 and S214, co-transfection of PPP3CA reversed the effect of GSK3 and dose-dependently reduced the level of phosphorylated tau (Fig. 6a). In addition, PPP3CB, another separately expressed subunit of PPP3CA, reverses the effect of GSK3? And dose-dependently reduces the level of phosphorylated tau (Fig. 9). Regression analysis revealed that the level of p-tau (S202 / T205) was inversely correlated with the expression of PPP3CA (R = 0.892) (FIG. 6b). Additionally, we investigated whether the catalytic subunit of PPP3CA is necessary and sufficient to dephosphorylate tau (Fig. 6c). To this end, the inventors transfected the cell with a catalytic site deletion mutation of PPP3CA (PPP3CAdeltaCAT), confirming that no tau dephosphorylation occurred in the mutant. The data show that the catalytic activity of PPP3CA is indispensable for the dephosphorylation of p-tau. We also demonstrated that shRNA was used to demonstrate the loss of function of PPP3CA (Fig. 6d), thereby abolishing PPP3CA function, resulting in a marked increase in tau phosphorylation by GSK3 ?.

Example  8: PPP3CA  / PP2B CTE  In vivo animal models of tau Dephosphorylation  Adjustment.

In order to confirm that the decreased expression of PPP3CA affects the phosphorylation of tau in vivo , we used an adenovirus vector (AAV vector) containing shRNA for PPP3CA as the hippocampal region of the tau (P301L) transgenic mouse And the difference between control and tau phosphorylation after repeated weight loss induced head injury was compared (Figure 6e). Consistent with the cell model, the knockdown of PPP3CA using AAV-shPPP3CA. The immune response of PPP3CA in the hippocampal region of the rats was markedly reduced. (Fig. 6F). In addition, the immune response of p-tau (S202 / T205) was strongly increased in the dentate gyrus after exposure to head injury due to weight loss (Fig. 6g). The p-tau (S202 / T205) immunoreactivity was increased in the PPP3CA knockdown region using AAV-shPPP3CA, but p-tau (Ser202 / Thr205) was not detected after the AAV-shRNA control transduction (Fig. 6h) . Similarly, the immunoreactivity of p-tau (S199) was also increased in the hippocampal regions (CA1, CA3, DG) of PPP3CA knockdown using AAV-shPPP3CA in several weight loss induced head injury (Fig. 10).

Claims (20)

A pharmaceutical composition for the prevention or treatment of neurodegenerative brain diseases caused by traumatic brain injury, comprising a modulator that increases the expression or activity of Calcineurin.
The composition of claim 1, wherein the calcineurin is PPP3CA (catalytic subunit, alpha isozyme) or PP2B (Serine / threonine-protein phosphatase 2B). The composition according to claim 1, wherein the modulator for increasing the activity of calcineurin is any one selected from the group consisting of a compound specifically binding to PPP3CA or PP2B, a peptide, a peptide mimetic, an aptamer, an antibody and a natural product .
2. The composition of claim 1, wherein the modulator that increases expression of calcineurin is an expression vector comprising a nucleic acid encoding calcineurin or a base sequence encoding mRNA or calcineurin.
The composition according to claim 1, wherein the neurodegenerative disease caused by traumatic brain injury is a neurodegenerative brain disease caused by aggregation of Tau protein or increased phosphorylation of tau protein.
3. The composition of claim 2, wherein said PPP3CA or PP2B inhibits phosphorylation or aggregation of tau protein.
Treating the neural tissue-derived cells with a candidate substance for treating a neurodegenerative brain disease,
Measuring the expression or activity of Calcineurin in the cell, and
When the expression or activity of calcineurin in the sample treated with the candidate substance is increased over the expression or activity of calcineurin in the sample not treated with the candidate substance, the candidate substance is classified into a neurodegenerative brain disease Wherein the therapeutic agent for neurodegenerative brain disease is selected from the group consisting of:
8. The screening method according to claim 7, wherein the neural tissue-derived cells are neurons or glial cells.
The screening method according to claim 7, wherein the calcineurin is PPP3CA (catalytic subunit, alpha isozyme) or PP2B (Serine / threonine-protein phosphatase 2B).
The method according to claim 7, wherein the expression of PPP3CA or PP2B is measured by Western blot, Co-immunoprecipitation assay, ELISA, real-time RT-PCR, Wherein the detection signal is any one selected from the group consisting of electrophoresis, immunostaining, and fluorescence activated cell sorter (FACS).
8. The screening method according to claim 7, wherein the neurodegenerative brain disease caused by traumatic brain injury is neurodegenerative brain disease caused by aggregation of tau protein or phosphorylation of tau protein.
A composition for the diagnosis of neurodegenerative brain diseases caused by traumatic brain injury, comprising an agent capable of measuring inhibition of activity of calcineurin.
13. The composition of claim 12, wherein the calcineurin is PPP3CA (catalytic subunit, alpha isozyme) or PP2B (Serine / threonine-protein phosphatase 2B).
13. The diagnostic composition of claim 12, wherein said agent is an antibody or an aptamer capable of specifically binding to PPP3CA or PP2B.
13. The diagnostic composition according to claim 12, wherein the neurodegenerative disease caused by traumatic brain injury is a neurodegenerative brain disease caused by aggregation of Tau protein or phosphorylation of tau protein.
A kit for the diagnosis of neurodegenerative brain diseases caused by traumatic brain injury, comprising an agent for measuring the expression or activity of Calcineurin.
The diagnostic kit according to claim 16, wherein the agent to be measured is a primer or a probe for a calcineurin gene, or an antibody against a calcineurin protein.
17. The diagnostic kit according to claim 16, wherein the neurodegenerative disease is a neurodegenerative brain disease caused by aggregation of Tau protein or phosphorylation of tau protein.
A method for providing information for diagnosing neurodegenerative brain diseases caused by traumatic brain injury, comprising the step of measuring the inhibition of PPP3CA or PP2B activity.
20. The method according to claim 19, wherein when the activity of PPP3CA or PP2B is inhibited, it is judged that neurodegenerative brain disease caused by traumatic brain injury has occurred.

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