Autophagy and mitophagy-related extracellular mitochondrial dysfunction of cerebrospinal fluid cells in patients with hemorrhagic moyamoya disease – Scientific Reports

Study population

The study participants were derived from an ongoing prospective multicenter stroke database from March 2016 to February 2022 (http://1ksgh.org/)^18,19,20,21. MMD diagnosis was performed by referring to a previous report²². More specifically, adult patients (≥ 18 years) with typical MMD angiographic features unilaterally or bilaterally were initially enrolled. Then, the exclusion criteria were applied based on risk factors, such as atherosclerosis, congenital anomaly, and prothrombotic past medical history, according to a previous study²². CSF samples from MMD patients with intracranial malignancy, infectious meningitis, end-stage hepatic or renal disease, and pregnancy were also excluded¹⁶. The treatment protocol for patients with hemorrhagic MMD was as follows: (1) cerebral angiography was performed to confirm whether there was an aneurysm that could cause bleeding and if so, surgery was performed; and (2) if increased intracranial pressure (IICP) was observed, extraventricular drainage (EVD) or craniotomy and hematoma removal was performed as needed. In the intensive care unit, lumbar CSF drainage was usually performed for 5 to 7 days. CSF samples within 72 h were collected and stored for analysis. CSF samples from two patients with idiopathic hydrocephalus were used as the control group (Fig. 1A). Two investigators reviewed the medical records (e.g. age, gender, underlying diseases, smoking, familial history of MMD, and prior MMD-related symptoms) and radiological findings (e.g. hemorrhagic pattern, bleeding focus, and Suzuki stage)⁵. A good neurologic outcome was defined as a modified Rankin scale score (mRS) of 0 to 3 at 3 months after first bleeding⁵. Specific mRS scores associated with good outcomes include: 0, no symptoms; 1, no significant disability; 2, slight disability, but able to look after own affairs without assistance; and 3, moderate disability that requires some help, but able to walk unassisted. This study was approved by the Institutional Review Board (No. 2017-9, 2018-6, and 2019-6) of the of the Hallym University-Chuncheon Sacred Hospital. All methods were performed in accordance with the Chuncheon Sacred Hospital guidelines and regulations. Informed consent was obtained from the patients or their relatives. Study was performed according to the STROBE guidelines.

Fluorescence-activated cell sorter analysis

Fluorescence-activated cell sorter analysis (FACS) was performed to identify extracellular mitochondria in the CSF samples (BD FACSCalibur, Becton–Dickinson, San Jose, CA, USA), according to our previous reports^17,18. A 100-µl CSF sample was stained with 200 nM MitoTracker Red CMXRos (Thermo Fisher Scientific, Waltham, MA, USA) for 40 min at room temperature. The potential cellular origin of the extracellular mitochondrial signals was evaluated using the following fluorescence-conjugated 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 (Miltenyi Biotec) (Supplementary Table S1). The CSF samples were immunostained with the antibodies for 20 min at room temperature and analyzed by flow cytometry. The data were analyzed using Flow Jo software (v 10.7.1 Ashland, OR, USA)^17,18.

Mitochondrial membrane potential

MMP was measured in the CSF cells using a MitoProbe JC-1 assay kit (Thermo Fisher Scientific)^16,17,18,23. JC-1 monomers emit green fluorescence (excitation/emission, 485/516). When they enter the mitochondria, JC-1 forms complexes known as J-aggregates, emitting high red fluorescence intensity from red aggregates (excitation/emission, 570/595). The JC-1 ratio of red to green fluorescence is generally used as a parameter to trace MMP. In brief, human CSF samples (100 µL) were incubated with 1 µL of 1 µM in dimethyl sulfoxide of JC-1 for 25 min at 37 °C in the dark. The fluorescence intensity was measured using GloMax Explorer (Promega, WI, USA). The MMP ratio was estimated by dividing the red fluorescence value by the green value.

Field emission transmission electron microscopy

CSF samples were centrifuged (2500 rpm, 5 min, room temperature) and examined by field emission electron microscopy (FE-TEM) by referring to previous protocols^17,18. FE-TEM was used to identify autophagic vacuoles and morphological changes in mitochondria in the CSF cells¹⁸. The pellets were fixed overnight in 2% glutaraldehyde in cacodylate buffer (0.1 M sodium cacodylate, 2 mM MgCl2, pH 7.4) at 4 °C. After washing three times with cacodylate buffer at 4 °C, the samples were post-fixed in 2% osmium tetroxide for 1 h at 4 °C. The samples were rinsed with deionized water and dehydrated in a graded ethanol series of 50% to 100% for 20 min at each step. The samples were incubated with progressively more concentrated propylene oxide dissolved in ethanol, followed by infiltration with increasing concentrations of Eponate 812 resin. The samples were dried in a 65 °C oven overnight and sectioned using an ultramicrotome. The sections were observed with an FE-TEM unit (JEM-2100F, JEOL) at the Korean Basic Science Institute (Chuncheon, Korea)^17,18.

Quantitative real-time reverse transcription analysis

We measured the mRNA expression of autophagy and mitophagy-related markers, including hypoxia-inducible-factor 1α (HIF1α), autophagy-related gene 5 (ATG5), pBECN1, BECN1 (ATG6), Bcl-1 antagonist X (BAX)/Bcl-2 ratio, BCL2 interacting protein 3 like (BNIP3L), death-associated protein kinase (DAPK)-1, and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1)^17,18. Total RNA was isolated from the CSF cells using the easy-BLUE RNA extraction kit (iNtRON Biotechnology, Korea) according to the manufacturer’s instructions. 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 2 × Rotor-Gene SYBR Green qPCR Master Mix (Qiagen, Carlsbad, CA, USA) in the Rotor-Gene Q (Qiagen). The PCR primers used in this study are presented in Supplementary Table S2.

Western blotting analysis

Western blotting was performed as in our previous studies^17,18. CSF cells were lysed with 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 of the supernatant were quantified using the BCA protein assay kit (Thermo Scientific). Equal amounts of protein were separated by 10 and 15% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). After blocking the PVDF membranes with a double blocker (phosphate-buffered saline buffer-based BDP) for 1 h, the transferred PVDF membranes were incubated with the primary antibodies overnight at 4 °C. After washing in Tris-buffered saline containing 0.1% Tween-20 (TBS-T buffer), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and developed using an enhanced chemiluminescence (ECL) Western blotting substrate kit (Thermo Fisher Scientific). The antibodies used in this study are presented in Supplementary Table S3.

Statistical analysis

Continuous variables are presented as the mean and ± standard deviation (SD). The chi-squared or Student’s t test was used to compare differences in the variables. Western blot reactions were quantified by estimating the optical density relative to that of actin used as the reference value.

The degree of qRT-PCR and western blots were described as the mean ± standard error of the mean (SEM). Statistical analysis was performed with SPSS V.21 (SPSS, IL, USA) and GraphPad Prism software (v.6.01; GraphPad Software Inc., San Diego, CA, USA). Statistical significance was indicated at p-values of < 0.05. P-values of less than of < 0.05 and 0.01 are represented by * and **, respectively.