KR20220060718A - DAPK1 biomarker for the diagnosis of delayed cerebral ischemia - Google Patents

Abstract

Related are a composition for diagnosis of delayed cerebral ischemia including a formulation capable of measuring an increased level of DAPK1 in cerebrospinal fluid (CSF) cells, a kit for diagnosis of delayed cerebral ischemia, and an information providing method for diagnosis of delayed cerebral ischemia. Accordingly, the present invention can be used for developing diagnostic kits specific for the occurrence of delayed cerebral ischemia when patients with subarachnoid hemorrhage are treated.

Description

DAPK1 biomarker for the diagnosis of delayed cerebral ischemia

The present invention relates to a composition for diagnosing delayed cerebral ischemia, a kit for diagnosing delayed cerebral ischemia, and a method for providing information for diagnosing delayed cerebral ischemia, which include a preparation capable of measuring an increased level of DAPK1 in cerebrospinal fluid cells (CSF).

Subarachnoid hemorrhage (SAH) is defined as acute hemorrhage in the subarachnoid space due to rupture of an intracranial aneurysm. Despite advances in minimally invasive surgical management, the mortality rate in patients with low SAH grade is still high, with a mortality rate of 24% for Hunt and Hess (H-H) grade 4 and 71% for grade 5. If the ruptured aneurysm is successfully treated with surgical clipping or endovascular coil embolization, the medical complications after SAH remain a difficult problem in the treatment of neurocriticality. Among complications, early brain injury (EBI) and delayed cerebral ischemia (DCI) are the main causes of poor neurological outcomes in patients with SAH. Early brain injury (EBI), blood-brain barrier and cerebral edema due to increased intracranial pressure and decreased cerebral perfusion pressure occur immediately after SAH ictus. Delayed cerebral ischemia (DCI) refers to a clinical syndrome characterized by a newly developed neurological deficit with fluctuating symptoms, usually occurring between 4 and 14 days after SAH ictus. DCI pathogenesis is known to be associated with cerebral vasospasm, cortical diffuse depolarization, microthrombosis, or EBI4, but the detailed mechanisms underlying DCI need to be further studied.

Korean Patent Publication No. 10-2019-0008216

The present invention provides a composition for diagnosing delayed cerebral ischemia comprising a preparation capable of measuring an increased level of DAPK1 in cerebrospinal fluid cells (CSF), a kit for diagnosing delayed cerebral ischemia comprising the composition, and diagnosis of delayed cerebral ischemia using the composition and kit The purpose is to provide a method for providing information for

On the other hand, the objects of the present invention are not limited to the above-mentioned objects, and other objects and advantages of the present application that are not mentioned can be understood by the following description, and will be more clearly understood by the examples of the present invention. . It will also be readily apparent that the objects and advantages of the present invention may be realized by the means and combinations thereof indicated in the appended claims.

A first aspect of the present invention is to provide a composition for diagnosing delayed cerebral ischemia, comprising a preparation capable of measuring an increased level of DAPK1 in cerebrospinal fluid cells (CSF).

A second aspect of the present invention is to provide a composition for diagnosing delayed cerebral ischemia, wherein the delayed cerebral ischemia occurs after subarachnoid hemorrhage.

A third aspect of the present invention is to provide a kit for diagnosing delayed cerebral ischemia comprising the composition described above.

A fourth aspect of the present invention is to provide an information providing method for diagnosing delayed cerebral ischemia, comprising measuring the increased level of DAPK1 in cerebrospinal fluid (CSF) cells isolated from a subject.

A fifth aspect of the present invention further comprises measuring the DAPK1 increased level of cerebrospinal fluid cells (CSF) isolated from the control and comparing the DAPK1 increased level of the cerebrospinal fluid cells (CSF) of the subject and the control. It is to provide an information providing method for diagnosing delayed cerebral ischemia.

According to the composition for diagnosing delayed cerebral ischemia of the present invention, a kit for diagnosing delayed cerebral ischemia comprising the composition, and a method for providing information for diagnosing delayed cerebral ischemia using the composition and kit, delayed cerebral ischemia, a severe complication occurring after subarachnoid hemorrhage This is the first finding that a significant elevation of DAPK1 in cerebrospinal fluid cells (CSF) is associated with the development of the disease. there is.

1 is a transmission electron microscope image of mitochondrial dysfunction with autophagy and mitophagy in CSF cells of SAH patients with DCI. Autophagic vacuoles containing damaged mitochondria in A and B are indicated by yellow arrows. Interactions between mitochondria and autophagic vacuoles in C and D are indicated by white asterisks. In E and F, autophagic vacuoles were observed near damaged mitochondria (red arrowheads) with matrix swelling and collapsed cristae. Mt, mitochondria; Av, fear of autophagy; N, nucleus. scale bar; 1 μm, 500 nm.
2 is an expression analysis of autophagy and mitophagy markers in CSF cells of a patient. In A, SAH patients with DCI had increased mRNA expression of DAPK1, BNIP3L and PINK1 than patients without DCI by qRT-PCR. Differences in mRNA expression of BAX, ULK1 and NDP52 were not statistically significant between the two groups. In B, increased protein expression of BECN at Ser15 and LC3II expression with phosphorylation and p62 degradation were observed in SAH patients with DCI. Actin was used as a loading control.
3 is a confocal microscope of mitochondrial dysfunction with increased autophagy and mitophagy in CSF cells from SAH patients with DCI. CSF cells positive for vWF were immunostained with antibodies specific for DAPK1, BNIP3L/NIX, PINK1 and BECN1 and analyzed microscopically. White dots indicated by yellow arrows indicate co-localization with autophagy and mitophagy markers in dysfunctional mitochondria labeled with red fluorescent.
Figure 4 Intracellular localization of DAPK1 in CSF cells of SAH patients with DCI. Expression and localization of DAPK1 were analyzed by immunogold labeling under EM. A, D, G, damaged mitochondria with edema are marked with an asterisk. Immunogold particles were mainly detected in mitochondria (white arrows). BC, EF, and HI, respectively, are boxed area enlargements of A, D, and G.
5 is a biological process of gene ontology (A) and protein-protein interaction network (B) of differentially expressed genes in relation to autophagy and mitophagy according to DC of the present invention, p62 is isolate 1 ( Also called SQSTM1).
6 is a gene ontology network of molecular functions (A) and cellular components (B) of differentially expressed genes related to autophagy and mitophagy in SAH patients with DCI, p62 is termed isolate 1 (SQSTM1).
7 shows the KEGG pathway of autophagy (A), and differentially expressed genes in relation to phagocytosis and mitophagy are shown in red, and p62 is also referred to as isolate 1 (SQSTM1). 8 is a KEGG pathway of mitophagy (B), and differentially expressed genes in relation to phagocytosis and mitophagy are indicated in red, and p62 is also referred to as isolate 1 (SQSTM1).

Hereinafter, the present invention will be described in detail. Prior to this, the terms or words used in the present specification and claims are not to be construed as being limited in their conventional meaning, and the inventor must properly understand the concept of the term in order to best describe his invention. Based on the principle that it can be defined, it should be interpreted as meaning and concept consistent with the technical idea of the present invention. Accordingly, the configurations described in the embodiments described in this specification are only the most preferred embodiment of the present invention, and do not represent all the technical spirit of the present invention, so various equivalents that can be substituted for them at the time of the present application It should be understood that there may be variations and variations.

According to an embodiment of the present invention, there is provided a composition for diagnosing delayed cerebral ischemia, comprising a preparation capable of measuring an increased level of DAPK1 in cerebrospinal fluid cells (CSF).

Autophagy is a restorative process that maintains cellular homeostasis through lysosomal degradation of dysfunctional cytoplasmic components. In the early stage after SAH, the autophagy pathway is activated, and an inappropriate or excessive autophagy response is closely related to the development of early brain injury (EBI). Meanwhile, we report a higher expression of beclin-1 (BECN1), microtubule-related protein light chain-3 (LC3) conversion and Cathepsin-D in the prefrontal cortex, suggesting an increased autophagy pathway in neurons after SAH. We also show that mitophagy is associated with oxidative stress-related neuronal cell death in a murine SAH model by endovascular perforation. Mitophagy is a process of maintaining healthy mitochondria by selectively removing damaged or dysfunctional mitochondria through an autophagy reaction. reported that knockdown of P phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) and parkin is associated with intracellular accumulation of mitochondrial fragments and damaged mitochondria via reduced mitophagy in endothelial cells. Mitochondrial dysfunction is associated with neuronal cell death in stroke pathogenesis, and mitochondrial depolarization in cerebral ischemic conditions increases reactive oxygen species (ROS), PINK1, and unfolding protein responses. There is a possible association between mitochondrial dysfunction and DCI development due to the decreased mitochondrial membrane potential in cerebrospinal fluid (CSF) cells observed in SAH patients with DCI. Given that DCI etiology can be more accurately assessed in CSF in the subarachnoid space surrounding cerebral arteries and aneurysms, we investigated mitochondrial dysfunction. Using autophagy and mitophagy in cerebrospinal fluid (CSF) cells to determine whether it can be reflected in the pathogenesis of DCI after SAH.

Reduced mitochondrial membrane potential in cerebrospinal fluid (CSF) was observed in subarachnoid hemorrhage (SAH) with delayed cerebral ischemia (DCI), but the mechanism of this association is unclear. Accordingly, the present inventors first evaluated whether mitochondrial dysfunction could be reflected in the etiology of delayed cerebral ischemia (DCI) by autophagy and mitophagy in cerebrospinal fluid (CSF) cells.

Hereinafter, the present invention will be described in more detail by way of Examples, but it is clear that the present invention is not limited by the Examples.

Experimental Example 1: Study Cohort

Induction cohorts were derived from regional stroke databases for March 2016 and May 2020. We included patients with SAH in this database with the following conditions: 1) adult patients 18 years of age or older; 2) SAH due to aneurysmal rupture; 3) dense local coagulation and/or vertical layers of blood greater than 1 mm thick on computed tomography (CT); and 4) SAH patients treated with endovascular coil embolization. Exclusion criteria were as follows: 1) non-aneurysmal SAH, such as trauma, infection, or pericerebral SAH, and 2) patients treated for surgical clipping. In this example, we enrolled only SAH patients with thick hemorrhage and underwent coil embolization, because initial hemorrhage and contamination of CSF by thin layer penetration during amputation or cerebral contractile injury may bias CSF mitochondrial outcomes. am.

Transmission electron microscopy TEM analysis was applied to detect mitochondrial autophagic vacuoles and morphological changes in CSF cells derived from SAH patients with DCI. We evaluated and compared autophagy and mitophagy biomarkers in cerebrospinal fluid (CSF) cells between patients with SAH with and without DCI using real-time quantitative PCR (qRT-PCR). Markers include death associated protein kinase DAPK-1, BCL2 Interacting Protein 3 Like (BNIP3L), Bcl-1 antagonist X (Bcl-1 antagonist X, BAX), phosphatase ) and tensin homolog (PTEN) induced kinase 1 (PINK1), nuclear dot protein 52 (NDP52) were included. We further evaluated the expression levels of the autophagy executor gene of BECN1 and the autophagy adapter protein of p62.20. Finally, we performed bioinformatics analysis to reveal interactions between differentially expressed genes in DCI pathogenesis.

DCI is defined as any of the following conditions, such as newly emerging neurological changes (e.g., ataxia, motor weakness, sensory changes, and decreased consciousness) and, if severe, on the Glasgow Coma Scale or with severe angiographic vasospasm. Scores of 2 or more on the National Health Agency Stroke Scale change. DCI was monitored daily using transcranial Doppler (TCD) velocity. When severe vasospasm was suspected in TCD (more than 200 cm/s in the middle cerebral artery or more than 85 cm/s in the basilar artery), catheter angiography was performed to additionally confirm the degree of vasospasm and chemical angioplasty. Poor outcome was defined as a modified Rankin Scale (mRS) score ≥ 3 or a Glasgow Outcome Scale (GOS) ≤ 4. This is because most of the differences in mitochondrial membrane potential of CSF cells were. Therefore, it was prominent at day 5 between SAH patients with and without DCI. We analyzed CSF samples obtained between days 5 and 7 with continuous lumbar drainage for investigation. This study was approved by the Institutional Review Board of participating hospitals (No. 2017-9, 2018-6, 2019-6) and informed consent was obtained from patients or their relatives.

Experimental Example 2: Transmission electron microscope

Previously, patients with SAH with DCI showed depolarization of mitochondrial membrane potentials, leading to changes in mitochondrial function and morphology. In the present invention, TEM was applied to investigate subcellular microstructure changes in CSF cells from SAH patients with DCI. CSF samples were centrifuged at 4000 rpm for 10 minutes and then pellets were examined under an electron microscope.

The pellet was fixed overnight in 2% glutaraldehyde in cacodylate buffer (0.1M sodium cacodylate, 2mM MgCl2) at 4°C. After washing 3 times with cacodylate buffer at 4°C, the samples were post-fixed in 2% osmium tetraoxide at 4°C for 1 hour. Samples were rinsed with deionized water and dehydrated through a gradient series of ethanol for 20 min for each step starting with 50% ethanol and ending with 100% ethanol. The samples were then incubated with progressively concentrated propylene oxide dissolved in ethanol and then infiltrated with increasing concentrations of Eponate 812 resin. The samples were baked overnight in a 65 oven and then sectioned using an ultramicrotome. The section was viewed with a Field Emission TEM unit (JEM-2100F, JEOL) in Chuncheon, Korea Basic Science Research Institute.

Experimental Example 3: Western Blot Analysis

CSF cells obtained from SAH patients were lysed with RIPA buffer (50 mM Tris-base, 10 mM EDTA, 150 nM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF). Protein lysates in the supernatant were quantified using a BCA protein assay kit (Thermo Scientific, USA). Equal amounts of protein were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Bio-Rad, USA). After blocking the membrane with 2% BSA in TBS-T (Tris-buffered saline containing 0.1% Tween-20) for 1 hour, the membrane was incubated with primary antibody at 4°C overnight. After extensive washing, membranes were incubated with HRP-conjugated secondary antibodies and developed using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, USA). Antibodies used in the present invention are BECN1 (Cell Signaling Technology, USA), p62 (Santa Cruz Biotechnology, USA), LC3 (Cell Signaling Technology, USA), pBECN1Ser15 (Cell Signaling Technology, USA), DAPK (Invitrogen, USA), PINK1 (Abcam, UK), BNIP3L/NIX (Abcam, UK), Actin (Santa Cruz Biotechnology, USA).

Experimental Example 4: Immunofluorescence staining

Multicolor immunofluorescence staining was performed using confocal microscopy to evaluate the colocalization of dysfunctional, mitochondrial autophagy and mitophagy producers in vWF-positive CSF cells. CSF cells were fixed with para-formaldehyde (4% w/v) and then washed with PBS. After blocking with 2.5% blocking solution (Vector, USA) for 1 h, cells were treated with DAPK1 (1:100), PINK1 (1;100), BECN1 (1;100) and BNIP3L/NIX (1:100) at 4 °C. left overnight After incubation with anti-rabbit Alexa Fluor 750-conjugated secondary antibody, coverslips were mounted on Fluoroshield® with DABCO (ImmunoBioScience Corp. USA). vWF-FITC (von Willebrand factor-fluorescein; 1:500, Abcam) was used as an endothelial marker to determine whether CSF cells were endothelial-derived. To evaluate dysfunctional mitochondria undergoing autophagy in vWF-positive CSF cells, autophagy- or mitophagy-related markers were identified as Mito Tracker Deep Red (MTDR, Invitrogen, USA) and co-immunofluorescence. The nucleus is DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride). Fluorescence images were obtained using a confocal microscope at 400x magnification.

Experimental Example 5: Real-time qRT-PCR

Given that loss of mitochondrial membrane potential leading to mitochondrial dysfunction leads to activation of mitochondrial autophagy, mitochondrial autophagy producers such as DAPK1, PINK1, BAX, BNIP3L, and NDP52 are CSF cells between SAH patients with and without DCI. was investigated in Total RNA was isolated from CSF cells using Trizol (Ivitrogen, USA) according to the manufacturer's instructions. cDNA was synthesized from 5 μg of RNA using Maxime RT PreMix Kit (iNtRON Biotechnology, Korea). The expression level of mitophagy-related genes was measured by qRT-PCR reaction in Rotor-Gene Q (Qiagen, USA) using 2x Rotor-Gene SYBR Green PCR Master Mix (Qiagen, Carlsbad, CA, USA). The primers were: DAPK1 (forward): 5'-GACCGTGAAGCATTACCTGAG-3' and (reverse) 5'-GCTGCTGAAGCTTTCCTTGTA-3'; BNIP3L (forward) 5'-ACAACAACAACTGC GAGGAAA-3' and (reverse) 5'-GAGGATGAGGATGGTACGTGT-3'; BAX (forward) 5'-GTTTCATCCAGGATCGAGCAG-3' and (reverse) 5'-CTGCAGCTCCATGTTACTG TC-3'; PINK1 (forward) 5'-GTATGAAGCCACCATGCCTAC-3' and (reverse) 5'-CATCATCTTGATGGCCAAGGG TC-3'; ULK (forward) 5'-AACAAGAAGAACCTCG CCAAG-3' and (reverse) 5'-CCGTT GCAGTACTCCATAACC-3'; NDP52 (forward) 5'-CCCCTACCATGGAGGAGACC -3' and (reverse) 5'-CCTGCAATTTCTGTCCTTGGC-3'

Experimental Example 6: Immunogold staining

We performed immunogold staining to identify the location of the most prominent differentially expressed markers within mitochondria in CSF cells from SAH patients with DCI. CSF cells were fixed with 0.1% glutaraldehyde and 2% paraformaldehyde in phosphate buffer at pH 7.4 at 4 °C for 1 h, followed by post-fixation with osmium tetraoxide at 4 °C for 30 min. Samples were dehydrated in graded series of ethanol. Samples were treated with a graded series of ethanol and embedded in LR white resin (EMS). The sections were then divided into ultra-thin sections at 80 nm and placed on a copper grid. For the aldehyde group without quenching, sections were treated with 0.02 M glycine for 10 min. Sections were then washed with deionized water, blocked in 1% BSA and incubated with rabbit anti-DAPK1 antibody (1:100) for 1 h. Grids were washed 5 times with 0.1% BSA in PBS and incubated in secondary antibody conjugated to 10 nm gold (AURION) diluted 1:100 in 0.1% BSA in PBS. The final samples were stained with uranyl acetate and lead citrate and then observed using a transmission electron microscope (JEOL-2100F, USA) at 200 KV.

Experimental Example 7: Bioinformatics analysis

Enhancement analysis was performed with visualization tools https://tools.dice-database.org/GOnet/ and https://www.genome.jp/kegg/tool/map_pathway2.html). Genes with an error detection rate of less than 0.05 were defined as significantly enriched. Additional protein-protein interaction (PPI) networks were performed using a search tool for the search of interacting genes/proteins (STRINGs). The minimum required interaction score was set to 0.7.31, the highest confidence score of 32.

Experimental Example 8: Statistical Analysis

Continuous variables are expressed as mean and ± standard deviation (SD). A chi-square or Student's t test was performed to identify significant differences between DCI and non-DCI patients. Comparison of qRT-PCR expression was performed using the Mann-Whitney U test test. Results were presented as median and 25-75th percentiles. Statistics were performed with SPSS V.21 (SPSS, Illinois, USA) and MedCalc (www.Medcalc.org) and statistical significance was expressed as p <0.05.

Evaluation Example 1: Clinical Features of the Study

A total of 57 SAH patients treated with endovascular coil embolization were included in the analysis (Table 1). Twenty-two patients (38.6%) experienced DCI during follow-up. There were no statistical differences in clinical outcomes (eg female sex, age, hypertension, diabetes mellitus, hyperlipidemia and smoking) between the two groups. SAH patients with DCI had higher Hunt and Hess grades IV and V, and had poor clinical outcomes at 3 months, but were not statistically significant. Because hypertension treatment was performed on DCI patients, the MAP for DCI was significantly higher than that for non-DCI patients (97.4 ± 7.7 mmHg for DCI versus 92.7 ± 3.6 mmHg for non-DCI). The number of anterior circulation aneurysms in DCI and non-DCI patients was 18 (81.8%) and 28 (80.0%), respectively.

variable Non-DCI (n=35) DCI (n=22) p-value Clinical Results female 22 (62.9%) 18 (81.8%) 0.1311 age 62.7 ± 17.1 57.6 ± 10.3 0.1623 High blood pressure 18 (51.4%) 9 (40.9%) 0.4428 diabetes mellitus 4 (11.4%) 2 (9.1%) 0.7814 hyperlipidemia 4 (11.4%) 4 (18.2%) 0.4788 smoking 4 (11.4%) 3 (13.6%) 0.8064 Hunt and Hess grade ≥ IV 11 (31.4%) 12 (54.5%) 0.0861 laboratory results hemoglobin 11.6 ± 1.2 12.0 ± 1.3 0.2891 SaO ₂ (%) 94.9 ± 1.6 95.2 ± 1.7 0.5909 MAP (mmHg) 92.7 ± 3.6 97.4 ± 7.7 0.0118 Heart rate (BPM) 89.6 ± 8.0 91.5 ± 6.2 0.3528 radiological findings Size (mm) 5.3 ±1.2 5.6 ±1.2 0.3671 forward circulation 28 (80.0%) 18 (81.8%) 0.8667 Treatment results chemical angioplasty - 9 (40.9%) poor clinical outcome 11 (31.4%) 10 (45.4%) 0.2895

BPM, beats per minute; DCI, delayed cerebral ischemia; MAP, mean arterial pressure. Data are presented as number of subjects (percentage) for discrete and categorical variables and mean ± standard deviation.

Evaluation Example 2: Morphological changes of mitochondria in SAH patients with DCI

TEM was applied to investigate changes in subcellular microstructure in CSF cells obtained from SAH patients with DCI (Fig. 1). Numerous autophagic vacuoles containing mitochondria and fusions of autophagic vacuoles with abnormal mitochondria with matrix edema and collapsed cristae accumulated in CSF cells. Autophagy vacuoles also appeared close to swollen mitochondria with inner mitochondrial membranes. These results indicated that mitochondrial dysfunction with morphological impairment was associated with autophagy flux, suggesting the possibility of autophagy and mitophagy in CSF cells in DCI pathogenesis.

Evaluation Example 3: Increase in mitochondrial autophagy in CSF cells

mRNA expression of autophagy and mitophagy markers in CSF cells was measured and compared between SAH patients with and without DCI. DCI patients exhibited significantly higher expression levels than non-DCI patients: DAPK1, 0.279 (0.227-0.293) in DCI versus 0.043 (0.021-0.086) in non-DCI, p < 0.0001; BNIP3L, 0.134 (0.060-0.199) in DCI versus 0.045 (0.020-0.101) in non-DCI, p = 0.0005; PINK1, 0.069 (0.044-0.820) in DCI versus 0.045 (0.012-0.063) in non-DCI, p = 0.006. BAX [0.208 (0.167-0.314), non-DCI 0.166 (0.095-0.223), p = 0.116], ULK1 [0.011 (0.003-0.025) versus 0.004 (0.001-0.008) non-DCI in DCI, p = 0.127] and NDP52 [DCI 0.237 (0.046-0.316) vs. [ratio DCI 0.155 (0.030-0.229), p = 0.123]. We further investigated autophagy markers, including ULK1, BECN, LC3 and p62, in CSF cells between DCI and non-DCI patients. Western blotting analysis revealed increased protein expression and phosphorylation of BECN1 at Ser15 in DCI patients compared to non-DCI patients (Fig. 2B). In addition, increased LC3II expression and p62 degradation were observed in DCI patients.

Evaluation Example 4: Autophagy and mitophagy of vWF-positive CSF cells in DCI

Previously, we demonstrated that cells of endothelial origin appearing to be vWF-positive are increasingly present in the CSF of DCI patients with reduced mitochondrial membrane potential. In SAH patients with DCI, multicolor immunofluorescence staining was performed using antibodies specific for vWF and MTDR as markers of dysfunctional mitochondria (Fig. 3). The fluorescence signals of DAPK1, BNIP3L/NIX, PINK1 and BECN1 overlapped with those of MTDRs in vWF-positive CSF cells. The results suggested that increased mitochondrial dysfunction due to autophagy and mitophagy in vWF-positive CSF cells may be associated with DCI pathogenesis in SAH patients.

Evaluation Example 5: Mitochondrial dysfunction with DAPK1 increase in DCI

Because DAPK1 expression was most significantly increased in DCI patients compared to non-DCI patients (Fig. 2A), we immunogold with anti-DAPK1 to confirm the enhanced subcellular localization of DPAK1 in CSF cells from SAH patients with DCI. Labeling was further performed (Fig. 4). Interestingly, DAPK-immune gold particles were mainly observed in damaged mitochondria with abnormal morphology, which is consistent with the results presented in Figure 3. Taken together, these results suggested that DAPK1 increase may play a pivotal role in mitochondrial dysfunction in DCI development.

Evaluation Example 6: Bioinformatics analysis

The top four categories of biological processes (BP) were positive regulation of autophagy, response to mitochondrial depolarization, positive regulation of apoptotic processes, and autophagy of mitochondria (Table 2 and Figure 5A). DCI-related molecular functions and cellular components were ubiquitin-like protein ligase binding, ubiquitin protein ligase binding, Lewy body and phagocytic assembly sites (Table 2 and Figure 6). PPI was the highest binding gene among autophagy and mitophagy markers with differential expression of BECN1, followed by BNIP3L, PINK1 and p62, also known as SQSTM1 (Fig. 5B).

ID GO term P value Genes biological process GO:0010508 positive regulation of autophagy 6.40E-09 BECN1, BNIP3L, DAPK1, PINK1 GO:0098780 response to mitochondrial depolarization 7.50E-09 BECN1, PINK1, SQSTM1* GO:0043065 positive regulation of apoptotic process 3.36E-08 BECN1, BNIP3L, DAPK1, PINK1, SQSTM1 GO:0000422 autophagy of mitochondrion 7.07E-08 BECN1, PINK1, SQSTM1 GO:0043066 negative regulation of apoptotic process 1.68E-07 BECN1, BNIP3L, DAPK1, PINK1, SQSTM1 GO:0016239 positive regulation of macroautophagy 3.70E-07 BECN1, BNIP3L, PINK1 GO:0000423 mitophagy 7.67E-07 BECN1,SQSTM1 GO:0007005 mitochondrion organization 1.17E-06 BECN1, BNIP3L, PINK1, SQSTM1 GO:1902902 negative regulation of autophagosome assembly 3.99E-06 BECN1, PINK1 GO:0033554 cellular response to stress 4.36E-06 BECN1, BNIP3L, DAPK1, PINK1, SQSTM1 GO:0016236 macroautophagy 5.14E-06 BECN1, PINK1, SQSTM1 GO:0010821 regulation of mitochondrion organization 7.32E-06 BNIP3L, PINK1, SQSTM1 GO:0034599 cellular response to oxidative stress 1.65E-05 BECN1, DAPK1, PINK1 GO:0006915 apoptotic process 2.07E-05 BECN1, BNIP3L, DAPK1, SQSTM1 GO:0060341 regulation of cellular localization 2.07E-05 BECN1, BNIP3L, PINK1, SQSTM1 GO:2000310 regulation of NMDA receptor activity 3.21E-05 DAPK1, PINK1 GO:0009057 macromolecule catabolic process 3.63E-05 BECN1, BNIP3L, PINK1, SQSTM1 GO:1903146 regulation of autophagy of mitochondrion 4.39E-05 BNIP3L, PINK1 GO:1903214 regulation of protein targeting to mitochondrion 4.82E-05 BNIP3L, PINK1 GO:0001666 response to hypoxia 4.91E-05 BECN1, BNIP3L, PINK1 GO:0002931 response to ischemia 5.99E-05 PINK1,SQSTM1 GO:0070887 cellular response to chemical stimulus 6.57E-05 BECN1, BNIP3L, DAPK1, PINK1, SQSTM1 GO:2000378 negative regulation of reactive oxygen species metabolic process 7.28E-05 BECN1, PINK1 GO:0051881 regulation of mitochondrial membrane potential 1.23E-04 BNIP3L, PINK1 GO:0031325 positive regulation of cellular metabolic process 1.25E-04 BECN1, BNIP3L, DAPK1, PINK1, SQSTM1 GO:1903827 regulation of cellular protein localization 1.86E-04 BNIP3L, PINK1, SQSTM1 GO:0044267 cellular protein metabolic process 2.35E-04 BECN1, BNIP3L, DAPK1, PINK1, SQSTM1 GO:0044257 cellular protein catabolic process 2.87E-04 BNIP3L, PINK1, SQSTM1 molecular function GO:0044389 ubiquitin-like protein ligase binding 3.62E-05 BECN1, PINK1, SQSTM1 GO:0031625 ubiquitin protein ligase binding 3.02E-05 BECN1, PINK1, SQSTM1 cellular component GO:0097413 Lewy body 1.43E-06 PINK1, SQSTM1 GO:0000407 phagophore assembly site 2.53E-05 BECN1, SQSTM1

The autophagy process of SAH has been studied in general for early brain injury (EBI). In neurons, autophagosomes and autolysosomes were increased up to 3 days after ictus. Autophagy activation peaked at 24 h and then recovered at 48 h. SAH rats treated with rapamycin, an autophagy activator that targets the mammalian target of rapamycin, showed reduced brain edema, BBB disruption, apoptosis, and improved behavioral measures. Therefore, it may be suggested that activation of the autophagy pathway may be a potential therapeutic target for alleviating EBI in the acute phase. In addition, we demonstrated the neuroprotective effect of mitophagy in ameliorating EBI via voltage-dependent anion channel (VDAC) 1 by reducing apoptotic and necrotic molecular pathways in SAH mice. We show epigallocatechin-3-gallate (EGCG) protective mitochondrial function by attenuation of cytoplasmic and mitochondrial Ca2+ concentrations, which in turn reduced depolarization of mitochondrial membrane potential, increased permeability of metastatic pore opening, and decreased ROS generation.

Compared with EBI, which occurs in the brain parenchyma during the first 72 hours after SAH onset, DCI usually occurs on the 3rd day thereafter and peaks between 7 and 14 days. SAH literally means acute hemorrhage within the subarachnoid space surrounding the cerebral artery. Free hemoglobin with subarachnoid space induces oxidative stress and inflammation, leading to DCI development. We first investigated the functional relevance of mitochondria in human CSF samples after SAH and in the present invention, higher mitochondrial membrane potential was reflected in favorable functional outcomes at 3 months follow-up. In particular, the higher the proportion of potentially occurring mitochondria in astrocytes, the better the results. With respect to DCI, it was found that mitochondrial membrane potential in CSF mitochondria at day 5 in SAH patients without DCI was significantly reduced compared to patients without DCI. A higher proportion of mitochondria of endothelial origin was observed more frequently in DCI patients compared to non-DCI patients. In the present invention, autophagy and mitophagy markers of cerebrospinal fluid (CSF) cells are further evaluated to elucidate the basis of DCI pathogenesis for mitochondrial dysfunction. In the present invention, cerebrospinal fluid (cerebrospinal fluid, CSF) cells showed that increased mitochondrial dysfunction due to autophagy and mitophagy could be responsible for the pathogenesis of DCI. Among the markers, DAPK1 was the most significant difference between DCI and non-DCI patients. In addition, immunogold particles with anti-DAPK1 antibody were mainly detected in mitochondria under EM.

사슬 상보 결정 영역 (CDR) 3 레퍼토리를 조사했다. 중증 DCI 환자는 CDR3 길이의 유의한 차이없이 중증이 아닌 DCI 환자에 비해 낮은 클론 성과 높은 다양성을 나타냈다. 따라서 CSF 공간에서 DCI 발병 기전에서 DAPK1 및 T 세포 면역의 역할에 대한 추가 연구가 더 필요하다. DAPK1 serves as a sensor of mitochondrial membrane potential in mitochondrial toxin-induced apoptosis. Mitochondrial toxins such as rotenone and the mitochondrial oxidative phosphorylation uncoupler CCCP (Carbonyl cyanide m-chlorophenylhydrazone) cause loss of membrane potential and mitochondrial expansion. It is a structure that leads to apoptosis through DAPK1 activation in neuroblastoma cells. Accumulation of PINK1 and LC3 on the outer membrane in damaged mitochondria is accompanied by a loss of membrane potential. Stroke-induced neuronal cell death is associated with DAPK1 signaling pathways such as DAPK1-N-methyl-D-aspartate receptor (NMDAR), DAPK1-p53. reported that under cerebral ischemia, DAPK1 directly binds to the NR2B subunit of NMDAR in the cortex of mice, which in turn enhances NR1/NR2B channel conductance, exacerbating calcium reflux. In addition, administration of NR2BCT, defined by a genetic deletion of DPAPK1 or a carboxyl tail region consisting of amino acids 1292-1304, inhibited calcium reflux and protected nerve damage after ischemic insult. DAPK-1 was also involved in the regulation of Activation of DAPK decreased T cell activation and IL-2 production. DAPK is a modulator for T cell receptor (TCR) activation NF-kappaB and T lymphocyte activation. Since DCI usually occurs on day 4 from SAH, T cell activation in the CSF may contribute to DCI. TCR in Peripheral Blood T Cells by High-throughput Sequencing in Patients with SAH

Three repertoires of chain complementarity determining regions (CDRs) were investigated. Patients with severe DCI showed lower clonality and higher diversity compared to patients with non-severe DCI without significant differences in CDR3 length. Therefore, further studies on the role of DAPK1 and T-cell immunity in DCI pathogenesis in the CSF space are needed.

Existing mitophagy is regulated by the PINK1/Parkin pathway. PINK1 remaining in the outer membrane in damaged mitochondria of autosomal recessive Parkinson's disease activates E3 ubiquitin ligase activity of parkin and recruits parkin. Selective lysosomal clearance was then followed by Parkin-mediated polyubiquitination. Parkin-mediated mitophagy was also involved in cardiac stress response mechanisms. Parkin-deficient mice showed increased mitochondrial dysfunction with decreased mitophagy myocardium. However, alternative Parkin-independent mitophagy or autophagy adapter proteins are also involved in the mitophagy pathway. Phosphorylation of Unc-51-like kinase 1 (Ulk1) in the heart under ischemic or glucose deprivation interacts with Ras-associated protein (Rab) 9. At serine 179, it forms a complex with Rip 1 and Dynamically Associated Protein (Drp) 1. Phosphorylation of Drp1 is then sequestered, followed by lysosomal degradation in mitochondria. PPI is the highest binding gene among autophagy and mitophagy markers in which BECN1 is differentially expressed between DCI and non-DCI patients. In addition, BECN1 actively participated in the autophagy and mitophagy pathways ( FIGS. 7 and 8 ). BECN1 is expressed within cytoplasmic structures such as mitochondria and endoplasmic reticulum and has three domains: a Bcl-2 homology motif, a coiled-coil domain and an evolutionarily conserved domain (ECD). BECN1 interacts with PI3KCIII/Vps34 via ECD and positively regulates autophagy. Diabetic mice showed increased autophagosome formation and autophagy flux with higher expression of BECN1, microtubule-associated protein light chain II (LC3-II) and lower expression of p62.49. For the treatment of cancer, BECN1 has been proposed as the following therapeutic target. Anti-tumor agents are used to enhance BECN1 activity. Functional enrichment analysis showed that positive regulation of autophagy and mitochondrial depolarization was most associated with biological processes in DCI development. Therefore, further studies of the control pathways regulating autophagy responses in mitochondria via BECN1 are needed to treat DCI after SAH.

In the present invention, we further evaluated confocal microscopy to identify autophagy and mitophagy markers differentially expressed in vWF-positive CSF cells from SAH patients with DCI. Mitochondrial dysfunction caused by autophagy and mitophagy may be actively involved in DCI pathogenesis. Endothelial dysfunction is associated with cerebrovascular disease. Nitric oxide and endothelin released from the endothelium regulate vascular function. Compared to healthy controls, SAH patients showed higher endothelial microparticles. Increased CSF and plasma endothelin levels were also observed in SAH patients with vasospasm. A higher proportion of vWF-positive mitochondria in DCI patients was observed compared to non-DCI patients. In TCD, patients with SAH with vasospasm showed significant increases in endothelial microparticles of CD105 + and CD62e + . Therefore, future studies should focus on the alleviation of the mitochondrial dysfunction of CSF cells resulting from the endothelium of DCI patients.

So far, the present invention has been focused on preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive point of view. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within an equivalent scope should be construed as being included in the present invention.

As described above, although the present invention has been described with reference to limited embodiments and drawings, the present invention is not limited thereto, and the technical idea of the present invention and the following by those of ordinary skill in the art to which the present invention pertains. Of course, various modifications and variations are possible within the scope of equivalents of the claims to be described.

Claims (5)

A composition for diagnosing delayed cerebral ischemia, comprising a preparation capable of measuring the increased level of DAPK1 in cerebrospinal fluid cells (CSF).
The method of claim 1,
The delayed cerebral ischemia will occur after subarachnoid hemorrhage, delayed cerebral ischemia diagnostic composition.
A kit for diagnosing delayed cerebral ischemia, comprising the composition of claim 1.
A method for providing information for diagnosing delayed cerebral ischemia, comprising measuring an increased level of DAPK1 in cerebrospinal fluid cells (CSF) isolated from a subject.
5. The method of claim 4,
measuring the DAPK1 increase level of cerebrospinal fluid cells (CSF) isolated from the control; and
Comparing the DAPK1 increase level of the cerebrospinal fluid cells (CSF) of the subject and the control; further comprising, information providing method for delayed cerebral ischemia diagnosis.

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