KR102466956B1 - DAPK1 biomarker for the diagnosis of delayed cerebral ischemia - Google Patents

Abstract

It 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, including an agent capable of measuring an increased level of DAPK1 in cerebrospinal fluid cells (CSF).

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, including an agent 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, mortality in patients with low SAH grades remains high, with mortality rates of 24% for Hunt and Hess (H-H) grade 4 and 71% for grade 5. Although ruptured aneurysms are successfully treated with either surgical clipping or endovascular coil embolization, medical complications following SAH remain challenging in neurocriticality therapy. Among complications, early brain injury (EBI) and delayed cerebral ischemia (DCI) are the main causes of poor neurological outcome 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 shortly after SAH ictus. Delayed cerebral ischemia (DCI) refers to a clinical syndrome characterized by newly developed neurological deficits with fluctuating symptoms, usually occurring between 4 and 14 days after SAH ictus. Although DCI etiology is known to be associated with cerebral vasospasm, cortical diffuse depolarization, microthrombosis or EBI4, the detailed mechanisms underlying DCI need to be further investigated.

Korean Patent Publication No. 10-2019-0008216

The present invention relates to a composition for diagnosing delayed cerebral ischemia comprising an agent 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 Its purpose is to provide a method of 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 not mentioned above 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 means of the instrumentalities and combinations indicated in the claims.

A first aspect of the present invention is to provide a composition for diagnosing delayed cerebral ischemia, including an agent capable of measuring the 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 above-described composition.

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 cells (CSF) isolated from a subject.

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

According to the composition for diagnosing delayed cerebral ischemia, the kit for diagnosing delayed cerebral ischemia comprising the composition, and the method for providing information for diagnosing delayed cerebral ischemia using the composition and kit of the present invention, delayed cerebral ischemia, a serious complication occurring after subarachnoid hemorrhage This is the first time that it has been found that a significant increase in DAPK1 in cerebrospinal fluid cells (CSF) is associated with the development, and the effect that can be expanded to the development of a diagnostic kit specific for the occurrence of delayed cerebral ischemia in the future treatment of patients with subarachnoid hemorrhage in the neurological intensive care unit is expected. have.

Figure 1 shows transmission electron microscopy images of mitochondrial dysfunction with autophagy and mitophagy in CSF cells from 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 with matrix swelling and collapsed cristae near damaged mitochondria (red arrowheads). Mt, mitochondria; Av, autophagic fear; N, nuclei. scale bar; 1 μm, 500 nm.
Figure 2 is an expression analysis of autophagy and mitophagy markers in the patient's CSF cells. In A, SAH patients with DCI showed 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. Increased protein expression of BECN at Ser15 in B and LC3II expression accompanied by phosphorylation and p62 degradation was observed in SAH patients with DCI. Actin was used as a loading control.
Figure 3 is a confocal micrograph of mitochondrial dysfunction with increased autophagy and mitophagy in CSF cells from SAH patients with DCI. vWF-positive CSF cells were immunostained with antibodies specific for DAPK1, BNIP3L/NIX, PINK1 and BECN1 and analyzed microscopically. White dots indicated by yellow arrows indicate colocalization with autophagy and mitophagy markers in red fluorescently labeled dysfunctional mitochondria.
Figure 4: Subcellular localization of DAPK1 in CSF cells of SAH patients with DCI. Expression and localization of DAPK1 was 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, HI, boxed area enlargements of A, D and G, respectively.
Figure 5 shows the biological process of the gene ontology (A) and protein-protein interaction network (B) of differentially expressed genes related to autophagy and mitophagy according to the DCs of the present invention, p62 isolate 1 ( It is also called SQSTM1).
Figure 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 referred to as sequestrant 1 (SQSTM1).
Figure 7 is the KEGG pathway of autophagy (A), and genes differentially expressed in relation to phagocytosis and mitophagy are marked in red, and p62 is also referred to as isolate 1 (SQSTM1). Figure 8 shows the KEGG pathway of mitophagy (B), and differentially expressed genes related to predation 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 this specification and claims should not be construed as being limited to a conventional dictionary meaning, and the inventor appropriately uses the concept of the term in order to describe his/her invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined. Therefore, the configuration described in the embodiments described in this specification is only one of the most preferred embodiments of the present invention, and does not represent all of the technical spirit of the present invention, so various equivalents that can replace them at the time of the present application It should be understood that there may be variations and variations.

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

Autophagy is a repair process that maintains cellular homeostasis through lysosomal degradation of dysfunctional cytoplasmic components. Autophagy pathways are activated in the early stages after SAH, and inappropriate or excessive autophagy responses are closely related to the development of early brain injury (EBI). Meanwhile, they reported higher expression of beclin-1 (BECN1), microtubule-related protein light chain-3 (LC3) turnover, and Cathepsin-D in the prefrontal cortex, suggesting an increased autophagic pathway in neurons after SAH. also show that mitophagy is associated with oxidative stress-related neuronal cell death in a rat SAH model by intravascular 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 was 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 under cerebral ischemic conditions, mitochondrial depolarization increases reactive oxygen species (ROS), PINK1 and unfolding protein responses. Reduced mitochondrial membrane potential in cerebrospinal fluid (CSF) cells observed in SAH patients with DCI makes a possible link between mitochondrial dysfunction and DCI development. Given that DCI etiology can be more accurately assessed in the 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 they may be reflected in DCI pathogenesis after SAH.

Reduced mitochondrial membrane potential in cerebrospinal fluid (CSF) has been 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 can be reflected in the pathogenesis of delayed cerebral ischemia (DCI) by autophagy and mitophagy in cerebrospinal fluid (CSF) cells.

Hereinafter, the present invention is further detailed through examples, but it is obvious that the present invention is not limited by the following examples.

Experimental Example 1: Study Cohort

The induction cohort was derived from regional stroke databases for March 2016 and May 2020. In this database, we included SAH patients with the following conditions: 1) adult patients aged ≥18 years; 2) SAH due to ruptured aneurysm; 3) dense focal coagulation and/or vertical layers of blood >1 mm thick on computed tomography (CT); and 4) SAH patients treated with endovascular coil embolization. Exclusion criteria were: 1) non-aneurysmal SAH, such as trauma, infection or pericerebral SAH, and 2) patients treated with surgical clipping. In this example, we enrolled only SAH patients with thick hemorrhage and underwent coil embolization, because contamination of the CSF by the initial hemorrhage volume and thin layer infiltration or brain shrinkage damage during amputation could bias the CSF mitochondrial results. to be.

Transmission electron microscopy TEM analysis was applied to detect mitochondrial autophagy 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 SAH patients 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 (BAX), phosphatase ) and tensin homolog (PTEN) induced kinase 1 (PINK1), nuclear dot protein 52 (NDP52). 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 new onset 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 points change on the National Institutes of Health Stroke Scale. DCI was monitored daily using transcranial Doppler (TCD) velocities. 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 additionally performed to 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 the difference in mitochondrial membrane potential of CSF cells was mostly. Thus, it became prominent on day 5 between SAH patients with and without DCI. We analyzed CSF samples obtained between days 5 and 7 through continuous lumbar drainage for our investigation. This study was approved by the Institutional Review Boards (No. 2017-9, 2018-6, 2019-6) of the participating hospitals, and informed consent was obtained from the patients or their relatives.

Experimental Example 2: Transmission electron microscope

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

Pellets were fixed overnight in 2% glutaraldehyde in cacodylate buffer (0.1 M sodium cacodylate, 2 mM MgCl2) at 4 °C. After washing three times with cacodylate buffer at 4°C, the samples were post-fixed in 2% osmium tetroxide for 1 hour at 4°C. Samples were rinsed with deionized water and dehydrated through a gradient series of ethanol with 20 min 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. Samples were baked overnight in a 65 oven and then sectioned using an ultramicrotome. Sections were viewed with a Field Emission TEM unit (JEM-2100F, JEOL) in Chuncheon, Korea Basic Science Institute.

Experimental Example 3: Western blot analysis

CSF cells obtained from SAH patients were lysed in 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 lysate in the supernatant was quantified using the BCA protein assay kit (Thermo Scientific, USA). Equal amounts of proteins were separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (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 the primary antibody overnight at 4°C. 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), and Actin (Santa Cruz Biotechnology, USA).

Experimental Example 4: Immunofluorescence staining

Multicolor immunofluorescence staining was performed using confocal microscopy to evaluate the colocalization of autophagy and mitophagy producers with dysfunctional, mitochondria in vWF-positive CSF cells. CSF cells were fixed with paraformaldehyde (4% w/v) and then washed with PBS. After blocking with 2.5% blocking solution (Vector, USA) for 1 h, cells were stained 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 in Fluoroshield ?? with DABCO (ImmunoBioScience Corp. USA). vWF-FITC (von Willebrand factor-fluorescein; 1:500, Abcam) was used as an endothelial marker to confirm that CSF cells were derived from the endothelium. To assess dysfunctional mitochondria undergoing autophagy in vWF-positive CSF cells, autophagy- or mitophagy-related markers are Mito Tracker Deep Red (MTDR, Invitrogen, USA) was co-immunofluorescence stained. The nucleus is DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride). Fluorescent 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 results in activation of mitochondrial autophagy, mitochondrial autophagy generators, such as DAPK1, PINK1, BAX, BNIP3L and NDP52, are important in 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 the Maxime RT PreMix Kit (iNtRON Biotechnology, Korea). Expression levels of mitophagy-related genes were measured by qRT-PCR reactions using 2x Rotor-Gene SYBR Green PCR Master Mix (Qiagen, Carlsbad, CA, USA) in Rotor-Gene Q (Qiagen, USA). Primers were as follows: DAPK1 (forward): 5′-GACCGTGAAGCATTACCTGAG-3′ and (reverse) 5′-GCTGCTGAAGCTTTCCTTGTA-3′; BNIP3L (forward) 5′-ACACAACAACTGC GAGGAAA-3′ and (reverse) 5′-GAGGATGAGGATGGTACGTGT-3′; BAX (forward) 5′-GTTTCATCCAGGATCGAGCAG-3′ and (reverse) 5′-CTTGCAGCTCCATGTTACTG 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 locate 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 for 1 hour at 4 °C, followed by post-fixation with osmium tetraoxide for 30 minutes at 4 °C. Samples were dehydrated in graded series of ethanol. Samples were treated with a graded ethanol series and embedded in LR white resin (EMS). The sections were then divided into ultra-thin sections at 80 nm and placed on copper grids. For aldehyde groups without quenching, sections were treated with 0.02 M glycine for 10 min. Sections were then washed in 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 observed using a transmission electron microscope (JEOL-2100F, USA) at 200 KV after staining with uranyl acetate and lead citrate.

Experimental Example 7: Bioinformatics analysis

Functional enrichment analysis was performed with visualization tools https://tools.dice-database.org/GOnet/ and https://www.genome.jp/kegg/tool/map_pathway2.html). Genes with a false discovery rate of less than 0.05 were defined as significantly enriched. An additional protein-protein interaction (PPI) network using a search tool for the search of interacting genes/proteins (STRING) was performed. The minimum required interaction score was set at 0.7.31, with the highest confidence of 32.

Experimental Example 8: Statistical Analysis

Continuous variables are expressed as mean and ± standard deviation (SD). Chi-square or Student's t tests were 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 expressed as medians and 25–75th percentiles. Statistics were performed with SPSS V.21 (SPSS, Illinois, USA) and MedCalc (www.Medcalc.org), and statistical significance was indicated by p < 0.05.

Evaluation Example 1: Clinical characteristics 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 a DCI during follow-up. There were no statistical differences in clinical outcomes (e.g. female gender, 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 poorer clinical outcomes at 3 months, but this was not statistically significant. Because antihypertensive treatment was performed on DCI patients, MAP in DCI was significantly higher than that in non-DCI patients (97.4 ± 7.7 mmHg in DCI versus 92.7 ± 3.6 mmHg in 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) DCIs (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 discovery Size (mm) 5.3 ±1.2 5.6±1.2 0.3671 anterior circulation 28 (80.0%) 18 (81.8%) 0.8667 treatment result 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 the subcellular ultrastructure in CSF cells obtained from SAH patients with DCI (Fig. 1). Numerous autophagic vacuoles containing mitochondria and fusions of abnormal mitochondria and autophagic vacuoles with matrix edema and collapsed cristae accumulated in CSF cells. Autophagy vacuoles also appeared close to swollen mitochondria with an inner mitochondrial membrane. These results indicated that mitochondrial dysfunction accompanied by morphological damage was associated with autophagic flux, suggesting the potential for autophagy and mitophagy in CSF cells in DCI pathogenesis.

Evaluation Example 3: Increased mitochondrial autophagy in CSF cells

The mRNA expression of autophagy and mitophagy markers was measured in CSF cells and compared between SAH patients with and without DCI. DCI patients showed 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, p = 0.127] and NDP52 [DCI 0.237 (0.046–0.316) versus [non-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 patients with DCI.

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

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

Evaluation Example 5: Mitochondrial dysfunction with DAPK1 increase in DCI

Since DAPK1 expression was most significantly increased in patients with DCI compared to patients without DCI (Fig. 2A), we immunogold with anti-DAPK1 to confirm enhanced subcellular localization of DPAK1 in CSF cells from SAH patients with DCI. Labeling was further performed (Fig. 4). Interestingly, DAPK immunogold 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 increased DAPK1 may play a pivotal role in mitochondrial dysfunction in DCI development.

Evaluation Example 6: Bioinformatics analysis

The top four categories of biological process (BP) were positive regulation of autophagy, response to mitochondrial depolarization, positive regulation of apoptosis process and mitochondrial autophagy (Table 2 and Fig. 5A). DCI-related molecular functions and cellular components were ubiquitin-like protein ligase binding, ubiquitin protein ligase binding, lewy bodies and phagocytic assembly sites (Table 2 and Fig. 6). PPI was the most highly associated gene among autophagy and mitophagy markers with BECN1 differentially expressed, followed by BNIP3L, also known as SQSTM1, PINK1 and p62 (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 processes 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 commonly studied in early brain injury (EBI). In neurons, autophagosomes and autolysosomes 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 can be suggested that activation of the autophagy pathway could be a potential therapeutic target to alleviate EBI in the acute phase. Furthermore, we demonstrated the neuroprotective effect of mitophagy in ameliorating EBI via voltage-dependent anion channel (VDAC) 1 by reducing apoptosis and necrosis molecular pathways in SAH rats. Demonstrate epigallocatechin-3-gallate (EGCG) protective mitochondrial function by attenuation of cytosolic and mitochondrial Ca2+ concentrations, which in turn reduced the depolarization of mitochondrial membrane potential, increased permeability of transitional pore opening and reduced ROS production.

Compared to EBI, which occurs in the brain parenchyma during the first 72 h after onset of SAH, DCI usually occurs on day 3 thereafter and peaks between days 7 and 14. SAH literally means acute hemorrhage within the subarachnoid space surrounding the cerebral arteries. Free hemoglobin in the subarachnoid space induces oxidative stress and inflammation, resulting in DCI development. We first investigated the functional relevance of mitochondria in human CSF samples after SAH and in our case, higher mitochondrial membrane potential was reflected in the favorable functional outcome at the 3-month follow-up. In particular, the higher the percentage of latently arising mitochondria in astrocytes, the better the results. Regarding DCI, mitochondrial membrane potential was found to be significantly reduced in CSF mitochondria at day 5 in SAH patients without DCI than in patients without DCI. A higher proportion of mitochondria of endothelial cell origin was more frequently observed 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 reveal the basics of DCI pathogenesis for mitochondrial dysfunction. In the present invention, cerebrospinal fluid (CSF) showed that increased mitochondrial dysfunction due to autophagy and mitophagy in CSF) cells could be responsible for DCI pathogenesis. 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 acts as a sensor of mitochondrial membrane potential in mitochondrial toxin-induced apoptosis. Mitochondrial toxins such as rotenone and carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial oxidative phosphorylation uncoupler, cause loss of membrane potential and mitochondrial swelling. 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 loss of membrane potential. Stroke-induced neuronal cell death is related to DAPK1 signaling pathways such as DAPK1-N-methyl-D-aspartate receptor (NMDAR) and DAPK1-p53. reported that under cerebral ischemia, DAPK1 directly binds to the NR2B subunit of NMDAR in mouse cortex, which in turn enhances NR1/NR2B channel conductance and exacerbates calcium reflux. In addition, administration of NR2BCT, defined by genetic deletion of DPAPK1 or the carboxyl tail region consisting of amino acids 1292–1304, inhibited calcium reflux and protected nerve damage after ischemic insult. DAPK-1 has also been involved in regulating: Activation of DAPK reduced T cell activation and IL-2 production. DAPK is a regulator for T cell receptor (TCR) activated 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 SAH patients

The chain complementarity determining region (CDR) 3 repertoire was investigated. Severe DCI patients showed lower clonality and higher diversity compared to non-severe DCI patients without significant differences in CDR3 length. Therefore, further investigation of the role of DAPK1 and T-cell immunity in DCI pathogenesis in the CSF space is warranted.

Conventional mitophagy is regulated by the PINK1/Parkin pathway. In damaged mitochondria of autosomal recessive Parkinson's disease, PINK1 remaining on the outer membrane activates Parkin's E3 ubiquitin ligase activity 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 myophagic 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) interacts with Ras-associated protein (Rab) 9 in ischemic or glucose-deprived hearts. At serine 179, it forms a complex with Rip 1 and dynamic 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 with BECN1 differentially expressed between DCI and non-DCI patients. BECN1 also 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 autophagic flux with higher expression of BECN1, microtubule-associated protein light chain II (LC3-II) and lower expression of p62.49. For cancer treatment, BECN1 has been proposed as a therapeutic target for cancer. Enhance BECN1 activity using antitumor agents. Functional enrichment analysis revealed that positive regulation of autophagy and mitochondrial depolarization were most relevant to biological processes in DCI development. Therefore, to treat DCI after SAH, further investigation of the control pathway regulating the autophagic response in mitochondria through BECN1 is needed.

In the present invention, confocal microscopy was further evaluated to identify differentially expressed autophagy and mitophagy markers in vWF-positive CSF cells of SAH patients with DCI. Mitochondrial dysfunction 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. Also, increased levels of CSF and plasma endothelin were observed in SAH patients with vasospasm. A higher proportion of vWF-positive mitochondria was observed in DCI patients compared to non-DCI patients. SAH patients with vasospasm in TCD showed significant increases in endothelial microparticles of CD105+ and CD62e+. Therefore, future studies should focus on the alleviation of mitochondrial dysfunction in CSF cells originating from the endothelium of DCI patients.

So far, the present invention has been mainly looked at with respect to preferred embodiments. Those skilled in the art to which the present invention belongs will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from a descriptive point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

As described above, although the present invention has been described by the limited embodiments and drawings, the present invention is not limited thereto, and the technical spirit of the present invention and the following by those skilled in the art to which the present invention belongs 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,
Including an agent capable of measuring the increased level of DAPK1 in cerebrospinal fluid (CSF) cells,
The delayed cerebral ischemia is a composition for diagnosing delayed cerebral ischemia, which occurs after subarachnoid hemorrhage.
delete A kit for diagnosing delayed cerebral ischemia, comprising the composition of claim 1.
As an information provision method for the diagnosis of delayed cerebral ischemia,
Measuring the increased level of DAPK1 in cerebrospinal fluid (CSF) cells isolated from the subject,
The delayed cerebral ischemia is an information providing method for diagnosing delayed cerebral ischemia, which occurs after subarachnoid hemorrhage.
According to claim 4,
Measuring the increased level of DAPK1 in cerebrospinal fluid (CSF) cells isolated from a control group containing healthy individuals; and
Comparing the level of DAPK1 increase in the cerebrospinal fluid (CSF) cells of the subject and the control group where the subarachnoid hemorrhage occurred; further comprising, information providing method for diagnosing delayed cerebral ischemia.

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