Diagnostic dapk1 biomarker for moyamoya disease and use thereof

Abstract

The present invention provides a method for predicting neurological prognosis based on mitochondrial dysfunction associated with autophagy and mitophagy by analyzing the characteristics and activity of extracellular mitochondria in cells present in cerebrospinal fluid (CSF) of adult patients with hemorrhagic MMD using a DAPK1 biomarker composition for predicting the prognosis of moyamoya disease comprising DAPK1. Measuring the number of mitochondria in cerebrospinal fluid (CSF) cells can be used to predict the prognosis of patients with Moyamoya disease, and autophagy and mitophagy markers expressed in these cells can be utilized in the development of therapies and treatments for the pathological mechanisms.

Description

Technology Field

[0001] The present invention relates to a DAPK1 biomarker for predicting the prognosis of Moyamoya disease and its use. Background Technology

[0002] Moyamoya disease is a cerebrovascular disease that occurs frequently, particularly among East Asians, and is characterized by a pathological phenomenon in which stenosis or occlusion progresses in the internal carotid artery, a major artery of the cerebrum, and in the proximal portions of its major branches, the anterior cerebral artery and the middle cerebral artery. At these sites of stenosis, new thin and abnormal blood vessels are formed compensatorily, forming a complex vascular network called the 'Moyamoya vessels,' which is interpreted as an attempt to maintain cerebral blood flow.

[0003] This disease manifests differently in children and adults; in children, it can cause transient symptoms due to cerebral ischemia or recurrent strokes, while in adults, there is a risk of it progressing to more severe cerebral hemorrhage. Since these symptoms can lead to neurological deficits or long-term complications in each age group, early diagnosis and appropriate treatment are crucial.

[0004] Currently, the cause of Moyamoya disease has not been clearly identified, but it is presumed that environmental factors, along with genetic elements, contribute to the pathology. While some studies have reported that specific gene mutations are associated with the disease, given the complexity of the disease, it appears that various genetic and non-genetic factors interact.

[0005] Diagnosis is primarily made through cerebral angiography, a method that can directly identify stenotic vessels and abnormal neovascularization, but entails high costs and certain risks. There is an urgent need for the development of non-invasive and cost-effective alternative diagnostic methods, and in particular, the introduction of biomarkers capable of early disease identification is expected to have a significant impact on disease management and prognosis improvement. This can contribute to a better understanding of the pathology of Moyamoya disease and the establishment of appropriate treatment strategies. Prior art literature

[0006] Korean Patent Publication No. 10-1974274 (Publication Date: April 30, 2019)

[0007] Korean Patent Publication No. 10-1810395 (Date of publication Dec. 20, 2017)

[0008] Korean Patent Publication No. 10-1493044 (Publication Date: Feb. 16, 2015) The problem to be solved

[0009] The present invention aims to solve the aforementioned problems. The present invention provides a method for predicting neurological prognosis based on mitochondrial dysfunction associated with autophagy and mitophagy by analyzing the characteristics and activity of extracellular mitochondria in cells present in cerebrospinal fluid (CSF) of adult patients with hemorrhagic MMD using a DAPK1 biomarker composition for predicting the prognosis of Moyamoya disease containing DAPK1.

[0010] The purposes of the present disclosure are not limited to those mentioned above, and other purposes and advantages of the present disclosure not mentioned may be understood from the following description and will be more clearly understood from the embodiments of the present disclosure. Furthermore, it will be readily apparent that the purposes and advantages of the present disclosure can be realized by the means and combinations thereof set forth in the claims. means of solving the problem

[0011] The present invention provides a DAPK1 biomarker composition for predicting the prognosis of Moyamoya disease comprising DAPK1.

[0012] The above DAPK1 may be a marker related to autophagy and mitophagy.

[0013] The present invention also provides a composition for predicting the prognosis of Moyamoya disease comprising a reagent capable of detecting the mRNA or protein expression level of DAPK1.

[0014] The agent for measuring the mRNA expression level may include an antisense oligonucleotide, primer, or probe that specifically binds to DAPK1.

[0015] The agent for measuring the protein expression level may include an antibody that specifically binds to DAPK1.

[0016] A kit for predicting the prognosis of Moyamoya disease comprising a composition is provided.

[0017] The above kit may be a DNA microarray chip or a protein chip.

[0018] The present invention also provides a method for providing information to predict the neurological prognosis of a patient with Moyamoya disease, comprising: a cell isolation step of isolating cells derived from a patient with Moyamoya disease; and a step of measuring the mRNA or protein expression level of DAPK1.

[0019] It may additionally include a mitochondrial number comparison step, which compares the number of mitochondria in a control group with a poor neurological prognosis with the number of mitochondria in a patient with hemorrhagic moyamoya disease.

[0020] In the cell separation step above, the cells may be cerebrospinal fluid cells (CSF cells).

[0021] The above step of comparing the number of mitochondria may involve comparing differences in mitochondrial expression through one or more analyses among flow cytometry, membrane potential analysis, and electron microscopy analysis.

[0022] In the mRNA or protein comparison step above, the expression level of mRNA may be measured by reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Realtime RT-PCR), RNase protection assay (RPA), or Northern blotting.

[0023] In the mRNA and protein comparison step above, the expression level of the protein may be measured by ELISA (enzyme linked immunosorbent assay), Western blot, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS (Fluorescence activated cell sorter), or microarray analysis. Effects of the invention

[0024] Measuring the number of mitochondria in cerebrospinal fluid (CSF) cells can be used to predict the prognosis of patients with Moyamoya disease, and autophagy and mitophagy markers expressed in these cells can be utilized in the development of therapies and treatments for the pathological mechanisms.

[0025] In addition, by using mitochondria derived from vascular cells in conjunction with other diagnostic methods utilizing radiology and clinical pathology, it can be very useful for predicting a more accurate prognosis and providing treatment tailored to individual symptoms.

[0026] In addition, by analyzing the mitochondrial status of individual patients, it supports the establishment of personalized treatment plans, which can contribute to maximizing treatment efficacy and minimizing side effects.

[0027] In addition, using cerebrospinal fluid through a non-invasive method can reduce the burden on patients and safely monitor the progression of the disease, thereby helping to maintain the patient's quality of life during treatment. Brief explanation of the drawing

[0028] Figure 1 is a graph showing the flow cytometry results of (A) the results of flow cytometry analysis of CSF cells of patients with Moyamoya disease and a control group, and (B) the differences in mitochondrial expression according to the neurological prognosis of patients with Moyamoya disease. Figure 2 is a graph showing the results of comparing (C) the difference in mitochondrial expression between poor and good prognoses in patients with moyamoya disease and (D) the difference in mitochondrial membrane potential. Figure 3 shows an electron microscope image of intracellular organelles of CSF cells to compare the number of mitochondria in cases of poor and good prognosis in patients with Moyamoya disease. Figure 4 shows a graph comparing the mRNA levels of CSF cells for autophagy and mitophagy markers by real-time PCR in cases of poor and good prognosis in patients with moyamoya disease. Figure 5 shows images of protein expression in CSF cells for autophagy and mitophagy markers in cases of poor and good prognosis in patients with moyamoya disease, confirmed by western blot. Figure 6 is a graph showing the results that, in the prognosis of a patient with Moyamoya disease according to an embodiment of the present invention, the number of mitochondria derived from blood vessel cells expressing von Willebrand factor significantly increased in the cases of poor and good. Specific details for implementing the invention

[0029] Definition of Terms

[0030] In this specification, “Moyamoya disease” refers to a chronic progressive cerebrovascular disease in which stenosis or occlusion is observed in the terminal portion of the intracranial internal carotid artery, that is, in the beginning portion of the anterior cerebral artery and middle cerebral artery, without any particular reason, and small abnormal blood vessels called Moyamoya vessels are observed in the vicinity. In one embodiment of the present invention, the Moyamoya disease may be characterized by developing due to a mutation in the RNF213 gene, but is not limited thereto.

[0031] In this specification, “autophagy” refers to a catabolic process that utilizes lysosomes to remove various cellular components, including unnecessary or denatured proteins, from within the cell. Furthermore, in addition to cancer and neurodegenerative diseases, autophagy is known to be associated with liver disease, heart disease, muscle disease, and pancreatic disease.

[0032] In this specification, “mitophagy” is an intracellular degradation mechanism that removes damaged or unnecessary mitochondria. It has been reported that when mitochondrial damage occurs, it forms a membrane-bound autophagosome and fuses it with a lysosome to selectively remove the damaged mitochondria. It is known that the activity of such mitophagy is important for regulating mitochondrial function and maintaining tissue function in various cells, including neurons.

[0033] Mitophagy is a selective mitochondrial degradation mechanism. Initially considered part of autophagy, which breaks down various intracellular elements for survival under nutrient-deficient conditions, it was later confirmed to be a mechanism capable of effectively removing damaged or unnecessary mitochondria by encapsulating them with a membrane, fusing them with lysosomes, and completely degrading them. Mitophagy plays a role in protecting mitochondria from the release of pro-apoptotic proteins, the generation of reactive oxygen species, and the hydrolysis of useless ATP from aging, damaged, and depolarized mitochondria.

[0034] In this specification, “biomarker” includes organic biomolecules such as peptides, polypeptides, nucleic acids, lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that exhibit a significant increase or decrease in gene or protein expression levels in individuals with a specific disease or disorder compared to a normal control group (individuals without the specific disease or disorder).

[0035] In the present specification, "vWF (von Willebrand factor)" is a blood glycoprotein related to hemostasis, also referred to as von Willebrand factor or von Willebrand factor. A deficiency or abnormality in the above vWF causes various diseases such as von Willebrand disease, thrombotic thrombocytopenic purpura, and Heyde's syndrome.

[0036] In one embodiment of the present invention, the vWF-positive mitochondria may be present outside the cell, specifically in the cerebrospinal fluid.

[0037] In one embodiment of the present invention, the vWF-positive mitochondria may be derived from an endothelial cell.

[0038] In one embodiment of the present invention, the composition comprises a preparation capable of confirming the origin of extracellular mitochondria present in cerebrospinal fluid, and specifically, may be a composition capable of confirming which cell the extracellular mitochondria originated from, but is not limited thereto.

[0039] In one embodiment of the present invention, the composition may include a preparation capable of detecting endothelial cell-derived mitochondria.

[0040] In one embodiment of the present invention, the mitochondria may represent one or more of extracellular mitochondria, mitochondrial particles, extracellular cerebrospinal fluid mitochondria, and extracellular mitochondrial particles.

[0041] In one embodiment of the present invention, the level of mitochondria may refer to the amount, number, concentration, etc. of the mitochondria, and specifically, the fact that the level of vWF-positive mitochondria of the individual is higher than that of the control group may mean that the number, concentration, or amount of vWF-positive mitochondria contained in the sample derived from the individual is greater than that of the control group.

[0042] In this specification, “to treat” or other forms of the word, e.g., “being treated” or “treatment,” means administering a composition or performing a method for reducing, preventing, inhibiting, disrupting, or eliminating a specific feature or event (e.g., vascular leakage). The treatment or prevention of a disease, condition, disorder, injury, or health problem may be partial or complete.

[0043] In this specification, the term “prognostic composition” means a mixture containing one or more compounds according to the present invention or physiologically / pharmaceutically acceptable salts or prodrugs thereof and other chemical components, and other components. Examples include physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism and to enable the expression of biological activity by facilitating the absorption of the active ingredient.

[0044] In this specification, “prognosis prediction” is used interchangeably with “prognosis” and refers to the act of predicting the course and outcome of a disease in advance. More specifically, prognosis prediction can be interpreted as any act of predicting the course of a disease after treatment by comprehensively considering the patient’s physiological or environmental condition, as the course of the disease after treatment may vary depending on the patient’s physiological or environmental state.

[0045] For the purposes of the present invention, the prognosis prediction described above may be interpreted as the act of predicting the disease-free survival rate or survival rate of a patient with Moyamoya disease by anticipating in advance the course of the disease and whether a cure is achieved after treatment of Moyamoya disease. For example, predicting that the "prognosis is good" implies that the patient's disease-free survival rate or survival rate after treatment is at a high level, meaning there is a high probability that the patient will be cured; conversely, predicting that the "prognosis is poor" implies that the patient's disease-free survival rate or survival rate after treatment is at a low level, meaning there is a high probability that the disease will recur or that the patient will die due to Moyamoya disease.

[0046] In this specification, “diagnosis” refers to any act capable of providing information related to said disease or disease, such as determining whether a specific disease or condition has occurred or the risk of its occurrence using the composition or kit of the present invention, as well as confirming the characteristics of a pathological state.

[0047] In this specification, "significant" or "significantly" refers to a statistically significant difference between values.

[0048] In this specification, “origin” may mean not only what is separated from a specific object or specific part, but also the object or part itself.

[0050] The present invention provides a DAPK1 biomarker composition for predicting the prognosis of Moyamoya disease comprising DAPK1.

[0051] The above DAPK1 may be a marker related to autophagy and mitophagy.

[0052] The present invention also provides a composition for predicting the prognosis of Moyamoya disease comprising a reagent capable of detecting the mRNA or protein expression level of DAPK1.

[0053] The agent for measuring the mRNA expression level may include an antisense oligonucleotide, primer, or probe that specifically binds to DAPK1.

[0054] The agent for measuring the protein expression level may include an antibody that specifically binds to DAPK1.

[0055] A kit for predicting the prognosis of Moyamoya disease comprising a composition is provided.

[0056] The above kit may be a DNA microarray chip or a protein chip.

[0057] The present invention also provides a method for providing information to predict the neurological prognosis of a patient with Moyamoya disease, comprising: a cell isolation step of isolating cells derived from a patient with Moyamoya disease; and a step of measuring the mRNA or protein expression level of DAPK1.

[0058] It may additionally include a mitochondrial number comparison step, which compares the number of mitochondria in a control group with a poor neurological prognosis with the number of mitochondria in a patient with hemorrhagic moyamoya disease.

[0059] In the cell separation step above, the cells may be cerebrospinal fluid cells (CSF cells).

[0060] The above step of comparing the number of mitochondria may involve comparing differences in mitochondrial expression through one or more analyses among flow cytometry, membrane potential analysis, and electron microscopy analysis.

[0061] In the mRNA or protein comparison step above, the expression level of mRNA may be measured by reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Realtime RT-PCR), RNase protection assay (RPA), or Northern blotting.

[0062] In the mRNA and protein comparison step above, the expression level of the protein may be measured by ELISA (enzyme linked immunosorbent assay), Western blot, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS (Fluorescence activated cell sorter), or microarray analysis.

[0064] Method for providing information to predict the neurological prognosis of patients with Moyamoya disease

[0065] The present invention includes a cell separation step, an expression level measurement step, and a mitochondrial number comparison step.

[0066] Step 1: Cell Separation Step

[0067] This is the step of isolating cells derived from Moyamoya disease patients.

[0068] During the cell separation stage, the cells may be cerebrospinal fluid (CSF) cells.

[0069] Step 2: Expression Level Measurement Step

[0070] This is a step to measure the mRNA or protein expression level of DAPK1.

[0071] In the expression level measurement step, the expression level of mRNA may be measured by reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Realtime RT-PCR), RNase protection assay (RPA), or Northern blotting.

[0072] In the expression level measurement step, the expression level of the protein may be measured using ELISA (enzyme linked immunosorbent assay), Western blot, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS (Fluorescence activated cell sorter), or microarray analysis.

[0073] Step 3: Mitochondrial count comparison step

[0074] This is the step of comparing the number of mitochondria in a control group with a poor neurological prognosis with the number of mitochondria in patients with Moyamoya disease.

[0075] It may involve comparing differences in mitochondrial expression through one or more analyses among flow cytometry, membrane potential analysis, and electron microscopy.

[0077] Experimental method

[0078] We investigated whether mitochondrial dysfunction in extracellular cerebrospinal fluid (CSF) related to autophagy and mitophagy may be involved in the neurological prognosis of adult patients with hemorrhagic moyamoya disease (MMD), for whom the pathogenesis associated with poor prognosis is not well understood.

[0079] CSF samples were collected from 43 adult MMD patients and analyzed according to results after 3 months. Mitochondrial cells and intact mitochondrial membrane potentials (MMPs) were evaluated using Fluorescence Activated Cell Classification System (FACS) and the JC-1 red / green ratio. Quantitative real-time polymerase chain reaction (ReTR) and Western blotting analyses were performed for autophagy and mitophagy-related markers, including HIF1α, ATG5, pBECN1, BECN1, BAX, BNIP3L, DAPK1, and PINK1. Finally, FACS analysis using specific fluorescent binding antibodies was performed to evaluate the potential cellular origin of CSF mitochondrial cells. Twenty-seven women (62.8%) with an average age of 47.4 ± 9.7 years were included in the study. Of the 43 patients with hemorrhagic MMD, 23 (53.5%) had a poor prognosis.

[0080] Differences in MMPs were evident between the two groups (2.4 ± 0.2 for patients with poor prognosis, 3.5 ± 0.4 for patients with good prognosis; p = 0.02). Significantly higher expression (2-ΔCt) of HIF1α, ATG5, DAPK1, BAX, and BNIP3L mRNA and proteins was also observed in patients with poor prognosis compared to those with good prognosis. A higher proportion of vWF-positive mitochondria suggestive of endothelial origin was observed in patients with good prognosis compared to those with poor prognosis (25.0 ± 1.4% for patients with good prognosis, 17.5 ± 1.5% for patients with poor prognosis; p < 0.01). We observed an association between increased mitochondrial dysfunction associated with autophagy and mitophagy in CSF cells and the neurological prognosis of adult patients with hemorrhagic MMD.

[0082] The present invention will be explained in more detail below through examples and experimental examples. However, these examples and test examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples and test examples.

[0083] Example 1: Flow cytometry analysis

[0084] Figure 1 is a graph showing the flow cytometry results of (A) the results of flow cytometry analysis of CSF cells of patients with Moyamoya disease and a control group, and (B) the differences in mitochondrial expression according to the neurological prognosis of patients with Moyamoya disease.

[0085] Study participants were derived from the ongoing prospective multicenter stroke database from March 2016 to February 2022.

[0086] More specifically, adult patients (18 years of age or older) with typical MMD angiographic features on one or both sides were initially enrolled. Then, exclusion criteria were applied based on risk factors such as atherosclerosis, congenital malformations, and a history of thrombosis.

[0087] CSF samples from MMD patients with intracranial malignancies, infectious meningitis, end-stage liver or kidney disease, or pregnancy were also excluded. The treatment protocol for hemorrhagic MMD patients is as follows: (1) Cerebral angiography was performed to check for aneurysms that could cause hemorrhage, and surgery was performed if present. (2) If increased intracranial pressure (IICP) was observed, extraventricular drainage (EVD) or craniotomy and hematoma removal were performed as needed.

[0088] In the intensive care unit, lumbar cerebrospinal fluid drainage was typically performed for 5 to 7 days. CSF samples were collected within 72 hours and stored for analysis. CSF samples from two patients with idiopathic hydrocephalus were used as a control group (Figure 1A). Two researchers reviewed medical records (e.g., age, sex, underlying diseases, smoking, family history of MMD, previous MMD-related symptoms) and radiological findings (e.g., hemorrhage pattern, hemorrhage focus, and Suzuki staging). A good neurological prognosis was defined as a modified Rankin scale score (mRS) of 0 to 3 at 3 months after the first hemorrhage.

[0089] Specific mRS scores associated with a good prognosis are as follows: 0 (no symptoms); 1 (no severe disability); 2 (slight disability but able to manage daily activities without assistance); 3 (moderate disability requiring some assistance but able to walk without assistance). Approval was obtained from the Institutional Review Board of Hallym University Chuncheon Sacred Heart Hospital (2017-9, 2018-6, 2019-6). All methods were performed in accordance with the guidelines and regulations of Chuncheon Sacred Heart Hospital. Informed consent was obtained from the patient or their relatives. The study was conducted in accordance with the STROBE guidelines.

[0090] Fluorescence-activated cell sorter analysis (FACS) was performed to identify extracellular mitochondria in CSF samples (BD FACSCalibur, Becton-Dickinson, San Jose, CA, USA).

[0091] 100 μl CSF samples were stained with 200 nM MitoTracker Red CMXRos (Thermo Fisher Scientific, Waltham, MA, USA) for 40 minutes at room temperature. The potential cellular origin of extracellular mitochondrial signals was evaluated using the following fluorescent binding antibodies: von Willebrand factor conjugated with fluorescein isothiocyanate (vWF-FITC, Abcam, Cambridge, MA, USA), glutamate aspartate transporter conjugated with allophycocyanin (GLAST-APC, Miltenyl Biotec, Madrid, Spain), CD45-FITC (Miltenyl Biotec), and CD41 / 61-FITC (Miltenyl Biotec).

[0092] CSF samples were immunostained with antibodies at room temperature for 20 minutes and analyzed by flow cytometry. Data were analyzed using Flow Jo software (v 10.7.1 Ashland, OR, USA).

[0094] Example 2: Mitochondrial Expression and Membrane Potential

[0095] Figure 2 is a graph showing the results of comparing (C) the difference in mitochondrial expression between poor and good prognoses in patients with moyamoya disease and (D) the difference in mitochondrial membrane potential.

[0096] MMPs were measured in CSF cells using the MitoProbe JC-1 assay kit (Thermo Fisher Scientific). The JC-1 monomer emits green fluorescence (excitation / emission, 485 / 516).

[0097] When JC-1 enters mitochondria, it forms a complex known as a J-aggregate, emitting high red fluorescence intensity from the red aggregate (excitation / emission, 570 / 595). The ratio of red to green fluorescence of JC-1 is commonly used as a parameter to track MMPs. Human CSF samples (100 μL) were incubated in the dark with 1 μM of JC-1 in 1 μL of dimethyl sulfoxide at 37°C for 25 minutes. Fluorescence intensity was measured using a GloMax Explorer (Promega, WI, USA). The MMP ratio was estimated by dividing the red fluorescence value by the green value.

[0098] Mitochondrial membrane potential is used as an important parameter of mitochondrial function. To measure extracellular mitochondrial membrane potential in cerebrospinal fluid, the MitoProbe JC-1 assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used according to the marker. The JC-1 monomer emits green fluorescence. When the monomer enters the mitochondria of healthy cells, JC-1 forms a complex called a J-aggregate and emits high red fluorescence. The red-to-green fluorescence ratio of JC-1 is primarily used as a parameter to monitor mitochondrial membrane potential. Based on this, the ratio of red-to-green fluorescence intensity was used to represent the mitochondrial membrane potential of the present invention. To measure the mitochondrial membrane potential, 100 μl of cerebrospinal fluid samples were first incubated with JC-1 (1 μl of 1 μM dimethyl sulfoxide) in a 96-well flat-bottom polystyrene plate (SPL, Pocheon, Korea) for 20 minutes at 37°C in the dark. The fluorescence intensities of the green monomer (excitation / emission 485 / 516) and red aggregate (excitation / emission 570 / 595) were measured using a GloMax explorer (Promega, WI, USA). The ratio was calculated by dividing the red fluorescence value by the green fluorescence value.

[0100] Example 3: Electron Microscope Observation

[0101] Figure 3 shows an electron microscope image of intracellular organelles of CSF cells to compare the number of mitochondria in cases of poor and good prognosis in patients with Moyamoya disease.

[0102] Cerebrospinal fluid (CSF) samples were centrifuged (2500 rpm, 5 min, room temperature) and examined using a field emission electron microscope (FE-TEM) in reference to the previous protocol.

[0103] FE-TEM was used to confirm mitochondrial autophagy vacuoles and morphological changes in CSF cells.

[0104] The pellet was fixed overnight at 4°C in 2% glutaraldehyde in cacodylate buffer (0.1 M sodium cacodylate, 2 mM MgCl2, pH 7.4). After washing three times with cacodylate buffer at 4°C, the sample was post-fixed in 2% osmium tetroxide at 4°C for 1 hour. The sample was rinsed with deionized water and dehydrated in a series of graded ethanol ranging from 50% to 100% for 20 minutes at each step. The sample was incubated with increasingly concentrated propylene oxide dissolved in ethanol, followed by infiltration with Eponate 812 resin at increasing concentrations. The sample was dried overnight in a 65°C oven and then cut using an ultramicrotome.

[0105] The cross-section was observed using the FE-TEM instrument (JEM-2100F, JEOL) at the Korea Basic Science Institute (Chuncheon).

[0107] Example 4: Real-time PCR Analysis

[0108] Figure 4 shows a graph comparing the mRNA levels of CSF cells for autophagy and mitophagy markers by real-time PCR in cases of poor and good prognosis in patients with moyamoya disease.

[0109] mRNA expression of autophagy and mitophagy-related markers, including hypoxia-inducing factor 1α (HIF1α), autophagy-related gene 5 (ATG5), pBECN1, BECN1 (ATG6), and Bcl-1 antagonist X (BAX) / Bcl, was measured. BCL2 interacting protein 3-like (BNIP3L), death-related protein kinase (DAPK)-1, and phosphatase and PTEN-induced kinase 1 (PINK1) were additionally measured.

[0110] Total RNA was isolated from CSF cells according to markers using the easy-BLUE RNA extraction kit (iNtRON Biotechnology, Korea). cDNA was synthesized from 1 μg of RNA using the Maxime RT PreMix Kit (iNtRON Biotechnology, Korea). mRNA expression was measured by quantitative real-time reverse transcription (qRT-PCR) analysis using the 2 × Rotor-Gene SYBR Green qPCR Master Mix (Qiagen, Carlsbad, CA, USA) from Rotor-Gene Q (Qiagen).

[0112] Example 5: Western blot analysis

[0113] Figure 5 shows images of protein expression in CSF cells for autophagy and mitophagy markers in cases of poor and good prognosis in patients with moyamoya disease, confirmed by western blot.

[0114] CSF cells were lysed in RIPA buffer (50 mM Tris base, 10 mM EDTA, 150 nM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein lysates in the supernatant were quantified using a BCA protein assay kit (Thermo Scientific) and separated by 10% and 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), after which they were transferred to a polyvinylidene fluoride (PVDF) membrane. Bio-Rad, USA) After blocking the PVDF membrane for 1 hour with a dual blocker (phosphate-buffered saline buffer-based BDP), the transferred PVDF membrane was incubated overnight with the primary antibody at 4°C. After incubating the membrane with a secondary antibody conjugated to horseradish peroxidase in buffered saline containing -0.1% Tween-20 (TBS-T buffer) at room temperature for 1 hour, an ECL (enhanced chemiluminescent) Western blotting substrate kit (Thermo Fisher Scientific) was used.

[0116] Example 6: Statistical Analysis

[0117] Figure 6 is a graph showing the results that, in the prognosis of a patient with Moyamoya disease according to an embodiment of the present invention, the number of mitochondria derived from blood vessel cells expressing von Willebrand factor significantly increased in the cases of poor and good.

[0118] Continuous variables were expressed as mean and ± standard deviation (SD). Differences in variables were compared using the chi-square or Student's t-test. Western blot reactions were quantified by estimating the optical density relative to the optical density of actin used as the reference value.

[0119] The results of qRT-PCR and Western blot were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using SPSS V.21 (SPSS, IL, USA) and GraphPad Prism software (v.6.01, Graph-Pad Software Inc., San Diego, CA, USA). Statistical significance was indicated at p-values ​​< 0.05. P-values ​​< 0.05 and < 0.01 are indicated as * and **, respectively.

[0120] Continuous variables were expressed as mean and ± standard deviation (SD). Data were analyzed using the chi-square or Student's t-test. To evaluate differences in JC-1 ratios based on the presence of delayed ischemia, repeated measures variation analysis (ANOVA), Mauchly's test of sphericity, and lower bound correction were performed. Additionally, JC-1 ratios and percentages according to specific mitochondrial types were compared using the Mann-Whitney U test. All statistical analyses were performed using SPSS V.21 (SPSS, Chicago, IL, USA) and MedCalc software (Medcalc, Mariakerke, Belgium), and a P-value < 0.05 was considered statistically significant.

[0122] Experimental Example 1: Clinical Characteristics

[0123] A total of 43 adult patients with hemorrhagic MMD, including 27 women (62.8%) with an average age of 47.4 ± 9.7 years, were enrolled for the post-exclusion analysis. Eight patients (18.6%) had a family history of MMD, and 14 (32.6%) had symptoms of ischemia prior to hemorrhage. The number of cases of unilateral MMD and posterior cerebral artery involvement was 9 (20.9%) and 9 (20.9%), respectively. Among the various types of ICH, IVH (n = 21, 48.8%), ICH with IVH (n = 10, 23.3%), and ICH (n = 9, 20.9%) occurred most frequently. Twenty-three patients (53.5%) received conservative treatment, and 20 received surgical treatment. EVD was performed on 13 patients, craniotomy and hematoma removal were performed on 7 patients, and 2 (2 / 7) of the patients underwent preoperative coil embolization to treat an aneurysm thought to be the cause of the hemorrhage, as shown in Table 1.

[0124] [Table 1]

[0125]

[0126] Three months after sporadic attacks, 23 patients (53.5%) had a poor neurological prognosis. The presence of IVH was observed more frequently in patients with a poor prognosis (n=18, 78.3%) than in patients with a good prognosis (n=13, 65.0%), but the difference was not statistically significant (p=0.334). Surgical treatment was also observed more frequently in patients with a poor prognosis (n=12, 52.2%), but the difference was not statistically significant (p=0.425). Overall, an analysis of clinical and radiological variables in adult patients with hemorrhagic MMD revealed that no variables were closely associated with patient prognosis at three months, as shown in Table 2.

[0127] [Table 2]

[0128]

[0130] Experimental Example 2: Flow cytometry analysis and mitochondrial expression, membrane potential results

[0131] Figure 1 is a graph showing the flow cytometry results of (A) the results of flow cytometry analysis of CSF cells of patients with Moyamoya disease and a control group, and (B) the differences in mitochondrial expression according to the neurological prognosis of patients with Moyamoya disease.

[0132] Figure 2 is a graph showing the results of comparing (C) the difference in mitochondrial expression between poor and good prognoses in patients with moyamoya disease and (D) the difference in mitochondrial membrane potential.

[0133] Flow cytometry analysis confirmed that more mitochondrial particles were present in patients with Moyamoya disease than in control patients.

[0134] According to FACS analysis, patients with hemorrhagic MMD exhibited more extracellular mitochondria in cerebrospinal fluid cells than the control group (Fig. 1A). Patients with a good prognosis had a significantly higher number of extracellular mitochondria in the cerebrospinal fluid than patients with a poor prognosis (Fig. 1B).

[0135] Regarding the prognosis of patients with Moyamoya disease, it was confirmed that there is a significant difference in mitochondrial expression between the Poor and Good groups.

[0136] In addition, it was confirmed that there is a significant difference in mitochondrial membrane potential.

[0137] Patients with a poor prognosis (2.4 ± 0.2) showed lower MMP levels according to the JC-1 red / green ratio compared to patients with a good prognosis (3.5 ± 0.4, p = 0.02) (Figure 2A and B).

[0139] Experimental Example 3: mRNA and protein expression results

[0140] Figure 3 shows an electron microscope image of intracellular organelles of CSF cells to compare the number of mitochondria in cases of poor and good prognosis in patients with Moyamoya disease.

[0141] Figure 4 shows a graph comparing the mRNA levels of CSF cells for autophagy and mitophagy markers by real-time PCR in cases of poor and good prognosis in patients with moyamoya disease.

[0142] Regarding the prognosis of patients with Moyamoya disease, a significant difference was confirmed by measuring the mRNA expression levels of the BAX biomarker in CSF cells for autophagy and mitophagy markers between the Poor and Good groups.

[0143] mRNA and protein expression of markers related to phagocytosis and mitophagy are shown in Figure 4. Compared to patients with a good prognosis, patients with a poor prognosis had significantly higher expression of HIF1α (2-ΔCt) (0.359 ± 0.069 vs. 0.581 ± 0.078; p = 0.041), ATG5 (0.024 ± 0.005 vs. 0.059 ± 0.014; p = 0.036), BAX (0.166 ± 0.031 vs. 0.355 ± 0.083; p = 0.047), BNIP3L (0.059 ± 0.017 vs. 0.147 ± 0.031; p = 0.022) and DAPK1 (0.012 ± 0.002 vs. 0.034 ± 0.010; p = 0.042). There was no significant difference between the two groups for other markers such as BECN1 and PINK1.

[0145] Experimental Example 4: Western blot analysis results

[0146] Figure 5 shows images of protein expression in CSF cells for autophagy and mitophagy markers in cases of poor and good prognosis in patients with moyamoya disease, confirmed by western blot.

[0147] Regarding the prognosis of patients with Moyamoya disease, a significant difference was confirmed by measuring the protein expression levels of the BAX biomarker in CSF cells for autophagy and mitophagy markers between the Poor and Good groups.

[0148] Western blotting analysis revealed that patients with a poor prognosis had significantly higher expression of HIF1α (0.456 ± 0.045 in good prognosis vs. 0.720 ± 0.057 in poor prognosis) and ATG5 (0.089 ± 0.006 in good prognosis vs. 0.532 ± 0.075 in poor prognosis), followed by DAPK1 (0.144 ± 0.070 in good prognosis, 0.468 ± 0.064 in poor prognosis), and BAX / Bcl-2 compared to those with a good prognosis.

[0150] Example 5: Statistical Analysis

[0151] Figure 6 is a graph showing the results that, in the prognosis of a patient with Moyamoya disease according to an embodiment of the present invention, the number of mitochondria derived from blood vessel cells expressing von Willebrand factor significantly increased in the cases of poor and good.

[0152] It was confirmed that patients with Moyamoya disease had a higher proportion of mitochondrial particles than control group patients.

[0153] FACS analysis was performed using specific fluorescent binding antibodies to evaluate the potential cellular origin of mitochondria in CSF cells.

[0154] Each antibody conjugate was evaluated for the following cell origins: vWF (endothelial cells), GLAST (astrocytes), CD41 / 61 (platelets), and CD45 (microglia / macrophages). Patients with a good prognosis had a significantly higher proportion of vWF-positive mitochondria (25.0 ± 1.4%) than patients with a poor prognosis (17.5 ± 1.5%; p < 0.01). GLAST (good prognosis 18.8 ± 1.5% vs. poor prognosis 15.2 ± 1.8%, p = 0.14), CD45 (good prognosis 16.5 ± 1.4% vs. poor prognosis 13.0 ± 1.5%, p = 0.11), and CD41 / 61 (good prognosis 10.3 ± 1.4% vs. poor prognosis 8.2 ± 1.1%, p = 0.22) did not differ significantly depending on the result, and the results are shown in Figure 6.

[0156] argument

[0157] Autophagy and mitophagy markers in cerebrospinal fluid cells were evaluated to elucidate the underlying mechanisms of neurological deterioration in adult patients with hemorrhagic MMD, focusing on mitochondrial dysfunction.

[0158] For the first time, it was demonstrated that cerebrospinal fluid mitochondria in adult patients with hemorrhagic MMD differ according to outcomes. First, patients with a good prognosis had a higher proportion of cerebrospinal fluid mitochondria with high MMP levels. Second, it was confirmed that increased mitochondrial dysfunction related to autophagy and mitophagy in cerebrospinal fluid cells contributes to neurological deterioration. Among the markers, the expression of HIF1α, ATG5, and DAPK-1 varied most significantly depending on the outcome. Third, a higher proportion of potentially endothelial-derived mitochondria was observed more frequently in adult hemorrhagic MMD patients with a good prognosis than in those with a poor prognosis.

[0159] MMD has been considered a model of chronic hypoperfusion. MCA specimens from MMD patients showed thicker intima-walls with increased HIF1α expression compared to controls.

[0160] HIF1α regulates oxygen homeostasis. The rate of hydroxylation decreases under ischemic conditions due to the generation of mitochondrial reactive oxygen species (ROS) or an inadequate O2 substrate.

[0161] Previous studies have reported that mitophagy is an adaptive metabolic response to hypoxia via a HIF1α-dependent pathway. In particular, it was confirmed that HIF1α-mediated responses reduce ROS levels and apoptosis caused by acute cellular exposure to ischemic diseases, and that mitochondrial HIF1α provides protection against oxidative stress.

[0162] In chronic cerebral ischemia caused by MMD, if ICH occurs, an acute cerebral ischemic state develops due to a sudden increase in intracranial pressure (IICP). Therefore, in adult patients with hemorrhagic MMD with a poor prognosis, an increase in HIF1α may be a compensatory process to protect against oxidative stress targeting mitochondria.

[0163] Autophagy and mitophagy regulate the inflammatory response in the CNS. In particular, the NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome is activated immediately after ICH.

[0164] Melatonin increased the expression of autophagy markers ATG5 and LC3-II / LC3-I and mitophagy markers Parkin and PINK-1, and NLRP3 inflammasome activation was found to decrease after SAH.

[0165] Conversely, autophagy inhibitors reversed these melatonin effects on autophagy, mitophagy, and the NLRP3 inflammasome. ATG5 plays an important role in both standard and non-standard autophagy processes. ATG5 has been reported to bind with ATG12 and ATG16 to form a complex, and it was confirmed that it was deposited on the autophagosome membrane after binding with LC3 and phosphatidylethanolamine (PE).

[0166] ATG5 is also involved in LC3-related phagocytosis. Through this process, ATG5 is involved in the fusion of autophagosomes and lysosomes.

[0167] CSF cells from patients with a poor prognosis show higher expression of ATG5 compared to patients with a good prognosis. The prognosis confirmed that autophagy function was enhanced to reduce ROS and neuroinflammation in patients with severe hemorrhagic MMD. Under ischemic reperfusion conditions, ATG5 knockdown reduced brain damage by decreasing excessive autophagy-induced ferroptosis.

[0168] DAPK1 is a regulator of autophagy and apoptosis and acts as an important component in mitochondrial toxin-induced apoptosis. Studies on the role of DAPK1 in MMD have rarely been conducted compared to neurodegenerative diseases or cancer. DAPK1 knockout mice were shown to have reduced inflammation and vascular damage in arterial hypertension-induced aneurysms.

[0169] Beclin1 also influences the action of DAPK1 on inflammation associated with aneurysm formation through the ATP-dependent NLRP3 inflammasome. DAPK1 was dephosphorylated, and its mRNA expression was activated in in vivo and in vitro models of cerebral ischemia, leading to increased binding of CypD to p53 translocated to mitochondria.

[0170] In addition, DAPK1 activation reduced MMPs along with mitochondrial edema in neuroblastoma cells. Previous studies confirmed that DAPK1 directly binds to the N-methyl-D-aspartate (NMDA) receptor NR2B subunit of Ser-1303, increasing NR1 / NR2B receptor channel conductivity and calcium influx in a stroke mouse model.

[0171] MMD blood vessels showed significant accumulation of hyaluronan and chondroitin sulfate in the thickened intima. It was also confirmed that hyaluronan synthase 2 is highly expressed in endothelial progenitor cells.

[0172] It was confirmed that changes in the perendothelial matrix of MMD vessels increase endothelial fragility against wall shear stress, which can lead to the excessive accumulation of hyaluronan and chondroitin sulfate in the endothelium of MMD vessels.

[0173] A higher proportion of vWF-positive mitochondria was observed in adult hemorrhagic MMD patients with a good prognosis compared to those with a poor prognosis. Migration of endothelial cells into the cerebrospinal fluid following the onset of ICH was observed in SAH patients but not in MMD. SAH patients showed higher synthesis of endothelial microparticles containing CD105+ and CD62e+ compared to the control group.

[0174] It was confirmed that DCI patients have a higher proportion of vWF-positive mitochondria in cerebrospinal fluid cells than non-DCI patients.

[0175] The RNF213 gene at 17q25, which codes for mysterin, represents the primary susceptibility gene for MMD. In particular, the p.R4810K mutation is associated with advanced MMD vascularization, posterior arterial circulation, and early age of onset.

[0176] Previous studies have reported a significant association with MMD patients compared to the control group [OR 48.1 (95% CI 29.1-79.6)]. The age of onset for MMD with GA and AA genotypes was younger than for MMD with GG genotype.

[0177] In addition, it was observed that the stroke recurrence rate was lower and the prognosis was better in MMD associated with GA and AA genotypes compared to GG genotype.

[0178] Protein mystery forms a cyclic oligomeric complex containing two serial AAA+ ATPase modules.

[0179] It was shown that genetic removal of mitochondrial matrix factors, including casein-degrading peptidase P (ClpP) and transcription factor Tfam, strongly induces the transcription of RNF213 in various organs, confirming that mysterin plays a role in MMD-like angiopathy through mitochondrial dysfunction and RNA dependence.

[0180] A higher proportion of vWF-positive mitochondria was observed in patients with a good prognosis compared to those with a poor prognosis in hemorrhagic MMD. In contrast, the number of vWF-positive mitochondria was increased in patients with SAH associated with DCI compared to patients without DCI. The expression of HIF1α, ATG5, and DAPK-1 showed maximum differences depending on the prognosis of adult hemorrhagic MMD patients, whereas in SAH, the differences in the expression of DAPK1, BNIP3L, and PINK1 were most pronounced. Differences in the degree of IVH and cellularity (ICH vs. IVH) may influence the statistical significance of prognosis. To determine the role of mitochondrial dysfunction in neurological prognosis following a hemorrhagic event in adult MMD, multivariate analysis of clinical and imaging variables and mitochondrial markers is essential.

[0181] In conclusion, the present invention demonstrated that extracellular mitochondria in CSF cells exhibit dynamic characteristics following hemorrhage in adult MMD patients. Mitochondrial dysfunction associated with autophagy and mitophagy in CSF cells may also be associated with the neurological prognosis of adult patients with hemorrhagic MMD.

[0182] In particular, autophagy and mitophagy-related markers of HIF1α, ATG5, and DAPK1 were significantly increased in patients with a poor prognosis compared to those with a good prognosis. Additionally, the number of cerebrospinal fluid mitochondrial cells likely derived from endothelial cells also varied significantly depending on the prognosis.

Claims

Claim 1 A DAPK1 biomarker composition for predicting the prognosis of Moyamoya disease, comprising a death-related protein kinase (DAPK)-1, wherein the death-related protein kinase (DAPK)-1 is derived from cerebrospinal fluid cells (CSF cells). Claim 2 A DAPK1 biomarker composition for predicting the prognosis of Moyamoya disease, characterized in that, in claim 1, the death-related protein kinase (DAPK)-1 is a marker associated with autophagy and mitophagy. Claim 3 A composition for predicting the prognosis of Moyamoya disease, comprising a reagent capable of detecting the mRNA or protein expression level of death-related protein kinase (DAPK)-1, wherein said death-related protein kinase (DAPK)-1 is derived from cerebrospinal fluid cells (CSF cells). Claim 4 A composition for predicting the prognosis of Moyamoya disease according to claim 3, characterized in that the reagent for measuring the mRNA expression level comprises an antisense oligonucleotide, primer, or probe that specifically binds to death-related protein kinase (DAPK)-1. Claim 5 A composition for predicting the prognosis of Moyamoya disease according to claim 3, characterized in that the reagent for measuring the protein expression level comprises an antibody that specifically binds to death-related protein kinase (DAPK)-1. Claim 6 A kit for predicting the prognosis of moyamoya disease comprising the composition of claim 3. Claim 7 A kit for predicting the prognosis of Moyamoya disease according to claim 6, characterized in that the kit is a DNA microarray chip or a protein chip. Claim 8 A method for providing information to predict the neurological prognosis of a patient with Moyamoya disease, comprising: a step of measuring the expression level of mRNA or protein expression of death-related protein kinase (DAPK)-1 from isolated cells derived from a patient with Moyamoya disease; wherein the cells are cerebrospinal fluid cells (CSF cells). Claim 9 A method for providing information to predict the neurological prognosis of a patient with Moyamoya disease, further comprising, in claim 8, a step of comparing the number of mitochondria of a control group with a poor neurological prognosis and the number of mitochondria of a patient with hemorrhagic Moyamoya disease. Claim 10 delete Claim 11 In claim 9, the mitochondrial number comparison step is characterized by comparing differences in mitochondrial expression through one or more analyses among flow cytometry, membrane potential analysis, and electron microscopy analysis, a method for providing information to predict the neurological prognosis of a patient with Moyamoya disease. Claim 12 A method for providing information to predict the neurological prognosis of a patient with Moyamoya disease, characterized in that, in the mRNA or protein measurement step, the expression level of mRNA is measured by reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Realtime RT-PCR), RNase protection assay (RPA), or Northern blotting. Claim 13 A method for providing information to predict the neurological prognosis of a patient with Moyamoya disease according to claim 8, wherein the expression level of the protein in the mRNA or protein measurement step is measured by ELISA (enzyme linked immunosorbent assay), Western blot, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS (fluorescence activated cell sorter), or microarray analysis.

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