Microglia-derived ADAM9 promote GHRH neurons pyroptosis by Mad2L2-JNK-caspase-1 pathway in subarachnoid hemorrhage

SAH patients

Forty cerebrospinal fluid (CSF) specimens and clinical data on age, sex, aneurysm location, aneurysmal size, Hunt & Hess score, Fisher grade, BMI, and GH were used in this study. Half of the specimens were used for mass spectrometry analysis, and the other half were used for the validation phase. Specimen and clinical data were collected from patients with acute nontraumatic SAH in the Department of Neurosurgery at Nanfang Hospital, Southern Medical University in Guangzhou, China, from September 2019 to August 2020. Patients who were admitted to the hospital within 24 h of ictus, presented with SAH, were diagnosed by initial brain computed tomography (CT) at onset, and were confirmed by digital subtraction angiography and/or computerized tomography angiography within 1–3 d of onset were included in the study. All patients were treated by endovascular coiling by a single cerebrovascular neurosurgeon. The study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) and approved by the medical ethics committee of Nanfang Hospital, Southern Medical University, and patients consented to the use of their specimens for the study.

Diagnosis of GHD

GHD was detected two weeks after the operation. The body mass index (BMI) and peak GH values after the insulin stimulation test were recorded for all patients. The diagnosis of GHD was defined as a peak GH value of less than 3 ng/mL after effective stimulation based on an insulin stimulation test or less than 11 ng/mL if the BMI was below 25 kg/m2, less than 8 ng/mL if the BMI was between 25 and 30 kg/m2 and less than 4 ng/mL if the BMI was greater than 30 kg/m2 according to an arginine + GHRH test [14].

Sample Preparation and Gel Electrophoresis

All CSF samples were obtained by lumbar puncture within 48 h of onset and placed in a 15 ml RNase/DNase-free centrifuge tube. The unfiltered CSF was centrifuged at 5000 × g at 4 °C for 10 min, and the supernatant was removed, transferred to 0.5 ml polypropylene tubes and stored at − 80 °C until further analysis. CSF samples were coded and analyzed in a blinded fashion. SDT lysis buffer was added to the sample. The lysate was sonicated and then boiled for 15 min. After centrifugation at 14,000 × g for 40 min, the supernatant was quantified with a BCA Protein Assay Kit (P0012, Beyotime). The sample was stored at − 20 °C. Twenty micrograms of protein from each sample was mixed with 6x loading buffer and boiled for 5 min. The proteins were separated on a 12.5% SDS–PAGE gel. Protein bands were visualized with Coomassie blue R-250 staining.

NanoLC–MS/MS (Nanoscale Liquid Chromatography coupled to Tandem Mass Spectrometry) analysis

The separated proteins were subjected to in-gel digestion before nanoLC–MS/MS analysis. The peptides were redissolved in solvent A (0.1% formic acid in water) and analyzed by online nanospray LC–MS/MS on an Orbitrap Exploris 480 coupled to an EASY-nLC 1200 system (Thermo Fisher Scientific, MA, USA). Two microliters of peptide sample was loaded on an analytical column (Thermo Fisher Scientific, Acclaim Pep Map RSLC 50 mm × 15 cm, nanoViper, P/N164943) and separated using a solvent B (0.1% formic acid, 80% ACN) gradient from 5 to 38% for 120 min. The column flow rate was maintained at 300 nl/min. An electrospray voltage of 2 kV relative to the inlet of the mass spectrometer was used. The mass spectrometer was run in data-independent acquisition mode and automatically switched between MS and MS/MS modes. The parameters were as follows: (1) MS: scan range (m/z) = 350–1500; resolution = 60,000; automated gain control (AGC) target = 3e6; maximum injection time = 50 ms; (2) higher-energy C-trap dissociation (HCD)–MS/MS: resolution = 30,000; AGC target = 1e6; collision energy = 28; and (3) data-independent acquisition (DIA) performed with a variable isolation window, with each window overlapping 1 m/z, a window number of 42, and a total cycle time of 3 s.

Protein identification and quantification

The raw DIA data were processed and analyzed by Spectronaut X (Biognosys AG, Switzerland) with the default settings, and the retention time prediction type was set to dynamic indexed retention time (iRT). Data extraction was determined by Spectronaut X based on extensive mass calibration. Spectronaut Pulsar X dynamically determines the ideal extraction window depending on the iRT calibration and gradient stability. The Q value (false discovery rate, or FDR) cutoff at the precursor and protein levels was set to 1%. Decoy generation was set to be mutated, which is similar to ‘‘scrambled’’ but applied with a random number of AA position swaps (min = 2, max = length/2). All selected precursors that passed through the filters were used for quantification. MS2 interference removes all interfering fragment ions except for the three least interfering ions. The average of the top three filtered peptides that passed the 1% Q value cutoff were used to calculate the major group quantities. Protein abundance was quantified by the area under the chromatographic peak of the MS peptide precursor ion signal intensity. Student’s t test was performed, and proteins were defined as differentially expressed if they had a p value < 0.05 and a fold change > 1.5.

Animals

A total of 565 male C57BL/6 mice (weight, 21 ± 2 g) were provided by the Laboratory Animal Center of Southern Medical University (Guangzhou, China). Mice had access to standard chow and water and were housed under standard laboratory conditions with a 12-h light–dark cycle at room temperature (RT) (24 ± 2 °C). All animal procedures were approved by the Animal Care Committee of Southern Medical University in accordance with the UK Animal Scientific Procedures Act 1986, the European Union (EU) Directive 2010/63/EU for animal experiments or the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 8023, revised in 1978). The study was approved by the Ethics Committee of Nanfang Hospital (ID: NFYY-2016-117). In addition, all efforts were made to minimize the number of animals used in the study and their suffering.

Mouse experiments

Endovascular perforation of the SAH was performed as we previously described [15]. Briefly, the mice were anesthetized, and a 5 − 0 nylon suture was advanced 3 mm past the bifurcation of the left internal carotid artery to induce arterial rupture, was withdrawn 3 mm and then readvanced 3 mm. In the sham group, the suture was not advanced past the internal carotid artery bifurcation. CSF was collected from the mice as previously described [16]. The sequences of primers used in the AAV were as follows: MAD2L2 (5′-CATCTTCCAGAAGCGCAAGAA-3′) and NC (5′-CAACAAGATGAAGAGCACCAA-3′).

Antibodies

The following primary antibodies were used: anti-ADAM9 (Thermo Fisher Scientific, Cat# PA5-76732, RRID: AB_2720459), anti-ADAM9 (Santa Cruz, Cat# sc-135822, RRID: AB_2221762), anti-Iba1 (Wako, Cat# 011-27991, RRID: AB_2935833), anti-CD11b (Abcam, Cat# ab184308, RRID: AB_2889154), GHRH (Thermo Fisher Scientific, Cat# PA5-92408, RRID: AB_2806480), GSDMD (Proteintech, Cat# 66387-1-Ig, RRID: AB_2881763), MAD2L2 (BD Biosciences Cat# 612266, RRID: AB_399583)), GFAP (Novus Cat# NBP2-33184DL594, RRID: AB_2923514), Olig2 (Proteintech, Cat# 66513-1-Ig, RRID: AB_2881876), NeuN (Abcam, Cat# ab104224, RRID: AB_10711040), caspase-1 (Abcam, Cat# ab207802, RRID: AB_2889889), GAPDH (Abcam, Cat# ab8245, RRID: AB_2107448), P-JNK (CST, Cat# 9255 S, RRID: AB_2307321), JNK (CST, Cat# 9252 S, RRID: AB_2250373), P-ERK (CST, Cat# 4370 S, RRID: AB_2315112), ERK (CST, Cat# 4695 S, RRID: AB_390779), P-p38 (CST, Cat# 9216 S, RRID: AB_331296), and p38 (CST, Cat# 8690 S, RRID: AB_10999090).

Tissue preparation for histology and immunostaining

Mice were euthanized using an i.p. injection of sodium pentobarbitone (1.6 mg/g BW), before being transcardially perfused with phosphate buffered saline (PBS) and 4% formalin. Brains were dissected, placed in formalin for 24 h, followed by PBS with 0.01% sodium azide. Tissue was cryoprotected in 30% sucrose with 0.05% sodium azide for 2 days. Free-floating serial brain Sect. (40 mm) were collected using a sliding microtome (Leica).

Immunofluorescence

For immunofluorescence staining of coronal frozen brain sections, the sections were rinsed with PBS, followed by blocking with 5% nonspecific-antigen goat serum for 1 h at 37 °C. Then, the sections were incubated overnight at 4 °C with specific primary antibodies. The next day, after rinsing with PBS containing 0.5% Triton X-100 three times, the sections were incubated for 1 h at 37 °C with the corresponding secondary antibodies conjugated to Alexa 488 or Alexa 594 (Thermo Fisher Scientific, USA). The primary and secondary antibodies were diluted in PBS containing 5% normal goat serum and 0.2% Triton X‐100. After incubation with secondary antibodies, the sections were mounted on glass slides, and cover slips were applied in mounting medium. Fluorescence images were captured with a confocal microscope (LSM880, Zeiss, Germany). For cell counts, the data are presented as the number of cells/mm2. For double-staining analysis, the data are presented as the ratio of the number of double‐positive cells to the number of single‐positive cells.

Morris water maze

The Morris water maze (MWM) test was performed as previously described [17]. Briefly, the first 5 d are dedicated to acquisition training. On day 6, the number of platform crossings and the time spent in the target quadrant were determined.

Open field test

The open-field arena test was performed using a Versamax animal activity monitor equipped with infrared photobeams as horizontal X–Y sensors and/or Z sensors. Mice were placed in the center of the open-field arena (40 cm × 40 cm × 30 cm) and allowed to explore for 60 min. The locomotor activity and location of the mice were scored automatically by VersaMax software. The percentage of time spent in the center area indicates anxiety levels.

Nissl staining

After the mice were perfused with 0.1 mol/L PBS followed by 4% paraformaldehyde (PFA), brain slices were removed, immersed in 4% PFA for 24 h and transferred to 30% sucrose solution until they sank. Subsequently, the brain slices were cut into 20 μm-thick transverse and horizontal sections using a freezing microtome (Thermo, USA). After the sections were incubated with 0.1% cresyl violet for 5 min at RT, the sections were rinsed in double distilled water followed by 95% ethanol, dehydrated in 100% ethanol, cleared in xylene, and covered in neutral resins. Images were acquired with a microscope (Nikon, Tokyo, Japan), and the neurons were counted with ImageJ software (Media Cybernetics, Bethesda, MD, USA).

Western blot

Western blot analyses were performed with an SDS‒PAGE system. Briefly, total extracted tissue was lysed with RIPA buffer containing protease inhibitor and protein phosphatase inhibitors at 4 °C for 30 min. The protein concentration was determined using a BCA assay kit (Beyotime Inc., China). Protein samples were separated using SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Then, the membranes were blocked with 5% BSA and incubated with primary antibodies overnight at 4 °C. The next day, after washing with TBST, the membranes were incubated with secondary antibodies for 1 h at RT. Finally, signals were detected using enhanced chemiluminescence reagents, and images were captured with a digital camera (Pierce, Rockford, IL, USA). The total gray value of each band was quantified with ImageJ software (NIH) and normalized to that of the loading control.

ELISA

ELISA was performed in accordance with the manufacturer’s protocol. The following kits and antibodies were used: a human ADAM9 ELISA kit (EHADAM9 × 10; Thermo Fisher; USA), an NPTX1 polyclonal antibody (20656-1-AP; Proteintech; USA), a YWHAB polyclonal antibody (10936-1-AP; Proteintech; USA), a mouse GH ELISA kit (EZRMGH-45 K; Millipore; USA), a mouse ACTH ELISA kit (ab263880; abcam; USA), a mouse PRL ELISA kit (ab214572; abcam; USA), a mouse TSH ELISA kit (EEL110; Thermo Fisher; USA), a mouse interleukin (IL)-1β ELISA kit (BMS6002TEN; Thermo Fisher; USA), a mouse IL-6 ELISA kit (E-EL-M0044; Elabscience; China), and a mouse IL-8 ELISA kit (abs520017; Absin; China).

Isolation and treatment of GHRH neurons and microglia

Mice were decapitated after inhalation anesthesia with isoflurane to obtain the whole brain. The hypothalamic ARC was dissected into single cells using successive incubations in 0.25% trypsin-EDTA. GHRH neurons were suspended, and microglia were adherent. The medium was replaced when the cells crawled out. For GHRH neurons, the medium was replaced with serum-free medium (DMEM/F12 containing 0.5% bovine serum albumin (BSA), B27, and N2 supplements in addition to 20 ng/mL bFGF and EGF). For microglia, freshly dissected cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were incubated at 37 °C and 5% CO2.

Single-cell RNA-seq analysis

Single-cell RNA-seq data and t-SNE projections were downloaded from https://tabula-muris.ds.czbiohub.org/; specifically, single-cell libraries from 100,605 cells were isolated from 20 organs from 3 female and 4 male 3-month-old C57BL/6JN mice. Gene count files were derived from cells prepared using the 10x Genomics platform and processed with CellRanger.

Statistical analysis

The results are expressed as the mean ± SD from at least three independent experiments and were analyzed by using GraphPad Prism 9 or SPSS 22.0 software (IBM, Armonk, NY, USA). Continuous variables with a normal distribution were compared using independent Student’s t tests, and those without a normal distribution were evaluated by nonparametric tests (Western blot, ELISA, and immunocytochemistry). In the MWM test, we averaged the escape latencies across the four trials for each mouse. These means were analyzed across five sessions. Two-way repeated-measures ANOVA was used for the main effect, with session as the repeated measure and escape latency as the dependent variable. A two-tailed probability value of < 0.05 was considered to indicate statistical significance. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

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