Cathepsin L-containing exosomes from α-synuclein-activated microglia induce neurotoxicity through the P2X7 receptor

Reagents and antibodies

Anti CTSL, anti Alix and anti P2X7R were purchased from Santa Cruz Biotechnology. Anti TSG101, anti CD63, anti α-synuclein and anti β-actin were purchased from Sigma-Aldrich (USA, St. Louis, Missouri). Anti AKT, anti p-AKT, anti caspase-3 and anti MAP2 were purchased from Cell Signalling Technology (USA). Anti CD11b and anti BSA were purchased from Abcam (UK). Anti CTSB was purchased from Proteintech (Wuhan, China). Purified human recombinant γ-Syn was purchased from absin (Shanghai, China) and endotoxin content of peptide was <0.1 ng/μg (1 IEU/μg) as determined by limulus amebocyte lysate (LAL) test determined at absin.

Lipopolysaccharides (LPS) from Escherichia coli 026:B6 were purchased from Sigma-Aldrich (USA, St. Louis, Missouri) and dissolved in H2O to create a 20 ng/μl of stock solution. LY294002 was purchased from Thermo Fisher (UK) and dissolved in dimethyl sulfoxide to create a stock solution of 25 mg/ml. BzATP, BBG and GW4869 were purchased from Sigma-Aldrich (USA, St. Louis, Missouri).

iCL was purchased from Enzo (USA) and it is a biomimetic material developed and synthesised based on E-64 and leupeptin, two natural inhibitors of CTSL. Studies have confirmed that CTSL and iCL have better adaptation in spatial structure, higher affinity, more specific binding and lower toxicity than natural inhibitors as well, and are widely used in vivo experiments in cells and animals57,58.


C57BL/6 mice purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) were used in this study. They were maintained on a 12-h light/dark cycle under standard conditions at a constant temperature (22 °C ± 2 °C) and humidity (50%–65%). Food and water provided ad libitum. All procedures involving animals were approved by the Shanghai Jiao Tong University School of Medicine Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978).

Primary cell culture

Primary cortical neurons were prepared from C57BL/6 mouse embryos on embryonic day 16–18 (E16-18). Pregnant mice were sacrificed under anaesthesia with sevoflurane, their embryos were recovered, and cerebral cortex tissues were collected from the embryonic brains. For neuron-enrichment, the meninges and blood vessels were carefully removed. The cerebral cortex tissues of the foetuses were removed, dissociated by mild trypsinization, and mechanically triturated in DNase solution (0.004% w/v) containing a soybean trypsin inhibitor (0.05% w/v). This mixture was centrifuged for 3 min at 1000 rpm, the supernatant was removed and the cell pellet was dissolved in neurobasal medium supplemented with 1% B-27 supplement (Thermo Fisher, UK), 5 mM l-glutamine, and 1% penicillin/streptomycin (PS; Thermo Fisher, UK). The cell suspension was seeded in 10 cm dishes and 12-well plates precoated with poly-d-lysine, and the plates were cultured in 95% air and 5% CO2 at 37 °C. The culture medium was replaced every 3−4 days. Neurons were used for experiments after 7 to 10 days in culture.

Primary mouse microglia were prepared from newborn C57BL/6 pups (1 day old) as described in a previous article4. Briefly, a total of 2 × 107–2.3 × 107 cells dissociated cells isolated from pooled neocortical tissues from male pups were plated in a 175 cm2 flask coated with poly-l-ornithine (Sigma-Aldrich, USA). After approximately 12–14 days in vitro, the flasks were shaken vigorously, and the medium was collected and centrifuged at 1000 rpm for 5 min to obtain a microglial pellet. For obtain purified exosomes from the cell culture supernatant, primary microglia were resuspended in DMEM/F-12 supplemented with 10% exosome-depleted FBS (SBI, USA) and 50 U/ml PS and separately seeded on poly-l-ornithine-coated coverslips in 12-well plates and 10 cm dishes for follow-up experiments. The cell purity was confirmed to be >98% by immunofluorescence using CD11b as a marker of microglia (Supplementary Fig. 2e).

To establish our cell model, purified human recombinant WT and A53T α-Syn (rPeptide, Athens, USA) were dissolved in ddH2O to create a 1 mg/ml stock solution and incubated at 37 °C in 5% CO2 for 7 days with constant agitation by using a mini Teflon stir bar to yield oligomeric α-Syn according to previous literatures59,60,61. Since potential contamination of endotoxin may confound the results of α-Syn on microglial activation, the amount of endotoxin in the purified α-Syn was determined at r-Peptide. The result showed that the content of endotoxin was <1.3 U/mg of peptide, which was consistent with previous studies and incapable of producing any significant neurotoxicity59,62.

Characterisation of purified α-synuclein aggregation

The procedures for protein oligomers negative-staining were established by TEM following our previously described methods with little modification63,64. Briefly, 5–10 μl of each sample was dropped on a carbon support membrane, followed by dropwise addition of 5–10 μl of 1% uranyl acetate. Then the membrane was dried at room temperature for testing. TEM images were collected with a Hitachi HT7700 by Shanghai Yuyi Technology Co., Ltd (Supplementary Fig. 2f).

Cell stimulation

Primary microglia were treated with LPS (100 ng/ml, E. coli, serotype 055: B5, Sigma-Aldrich, USA) or recombinant α-Syn oligomers (250 nM). Cells, cell supernatants and exosomes were collected for analysis of protein expression and other parameters (see below).

Isolation and purification of exosomes

The supernatants of primary microglia collected from each sample were centrifuged at 2000 × g for 30 min to remove cell debris and dead cells (Beckman Coulter, Allegra X-14R). Exosomes were then purified from the supernatants using an exosome isolation kit (Thermo Fisher, UK, Cat# 4478359). We added 0.5 volumes of the Total Exosome Isolation reagent into the required volume of cell-free culture media and then mixed the culture media/reagent mixture well by vortexing. After incubation at 4 °C overnight, samples were centrifuged at 10,000 × g for 1.5 h at 4 °C. The supernatant was aspirated and concentrated by a Macrosep Advance centrifugal device with a molecular weight cutoff of 1 kDa (Pall Life Sciences, MI) to prepare the exosome-depleted fraction, and the pellet containing exosomes at the bottom of the tube was resuspended in 150 μl RIPA buffer (Beyotime Biotechnology, China) to prepare the exosome-enriched fraction.

Exosome identification

A total of 5 μl exosome suspension was spread on a copper grid at room temperature for 1 min. Filter paper was then used to remove the superfluous liquid. Afterwards, the exosomes were negatively stained using 1% (w/v) sodium phosphotungstate solution at room temperature for 3 min, and the solution was removed with filter paper. Then, the copper grid was placed under a tungsten lamp for 10 min. For transmission electron microscopy (TEM) (De Jong Instruments, USA), isolated and purified exosomes were fixed in 2% glutaraldehyde and 2% paraformaldehyde, dried, mounted and coated with gold/palladium. A Flow Nano Analyser (NanoFCM Co., Ltd, China) was used to determine the diameter and size distribution of the exosomes.

Nanoparticle tracking analysis (NTA)

Exosome size and concentration were measured by using NTA with a ZetaView PMX 110 analyser (Particle Metrix, Meerbusch, Germany) and ZetaView 8.04.02 software. Briefly, isolated exosome samples were appropriately diluted using 1x PBS buffer (Biological Industries, Israel) to measure the particle size and concentration. NTA was performed at 11 positions. The ZetaView system was calibrated using 110 nm polystyrene particles. The temperature was maintained at approximately 25 °C to 26 °C.

PKH67 labelling of exosomes and lactate dehydrogenase (LDH) assay

Purified exosomes were labelled with a PKH67 green fluorescent labelling kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Briefly, exosome pellets obtained from 20 ml culture medium were resuspended in 500 ml Dilution C, mixed with PKH67 (2 ml) diluted in 500 ml Dilution C, and then incubated for 3 min at room temperature. An equivalent volume of 1% BSA was added to bind the excess PKH67. Exosomes labelled with PKH67 were collected as mentioned above, resuspended in neurobasal medium and added to neurons (cultured in 12-well plates). A Lactate Dehydrogenase (LDH) Assay Kit (Abcam, UK) was used to measure LDH levels in microglia to evaluate cellular damage after different treatments.

RNA interference and transfection

P2X7R and CTSL knockdown experiments were performed by using primary microglia seeded in 10 cm dishes at 60%–80% confluence. The cells were transfected with P2X7R, CTSL or control small interfering RNA (siRNA) (scramble) (GenePharma, Shanghai, China) by using Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher, UK) according to the manufacturer’s instructions. The siRNA sequences were as follows: P2X7R siRNA: GCACAGUGAACGAGUAUUATT, UAAUACUCGUUCACUGUGCTT; CTSL siRNA: GGGCCUAUUUCUGUUGCUATT, UAGCAACAGAAAUAGGCCCTT; control siRNA (scramble): UUCUCCGAACGUGUCACGUTT, ACGUGACACGUUCGGAGAATT.

Western blot analysis

Microglial medium was harvested, desalted, and concentrated by a Macrosep Advance centrifugal device with a molecular weight cutoff of 1 kDa (Pall Life Sciences, MI) to prepare cell supernatant samples. Microglial exosomes were isolated and purified by using an exosome isolation kit as described above. The protein concentration was determined by the Bradford protein assay. Proteins isolated from cell supernatants, whole-cell lysates and prepared exosomes were separated by SDS-PAGE (Millipore, Billerica, MA and Bio-Rad, Hercules, CA), the membranes were incubated overnight at 4 °C with primary antibodies. After washing, the membranes were incubated with horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibodies (1:20000, Jackson ImmunoResearch, USA) for 1.5 h at room temperature. The bands were visualised using chemiluminescence (BIO-RAD ChemiDoc MP Imaging System), and the density of each band was normalised to that of the loading control band, which was showed by silver staining using a ProteoSilver Silver Stain Kit (Sigma-Aldrich, USA). The loading control band was used to unify the loading quantity of exosome samples65. All blots were processed in parallel and derive from the same experiment.

Coimmunoprecipitation (co-IP)

The interaction between extracellular α-Syn (oligomer and monomer) and P2X7R was assessed as previously described4. Briefly, precleaning was performed by the addition of 50 μl rProtein G agarose beads (Thermo Fisher, UK) and 2 μg anti-P2X7R, anti-human α-Syn, or species-relevant nonspecific mouse or rabbit immunoglobulin G (IgG). After 4 h of rotation at 4 °C, the beads were centrifuged at 1000 × g for 3 min and then washed with PBS 4 times. Primary microglia were harvested and lysed with co-IP lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 10% glycerol, and 0.5% Triton X-100) supplemented with 1:100 Halt protease and phosphatase inhibitor cocktail (Roche, Switzerland). The lysates were rotated at 4 °C for 30 min and cleared by centrifugation at 12,000 × g. Samples were then incubated with antibody-conjugated beads overnight at 4 °C. The proteins were separated by SDS-PAGE (10–12%) and electrotransferred onto polyvinylidene fluoride membranes. Western blot analysis was carried out as described above.

CTSL activity assay

We harvested the supernatants of primary microglia and isolated exosomes in accordance with the above methods. CTSL activity was assessed by using an activity assay kit (Abcam, UK) according to the manufacturer’s instructions, and fluorometric intensity was measured in a white flat-bottom Costar 96-well plate (Corning, Lowell, MA) using a plate reader (AnalystTM AD 96-384, Biosystems, USA) at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Background readings were subtracted from the sample values.

Immunofluorescence analysis

To assess the localisation of CTSL and exosomes, and the colocalization between α-Syn oligomer and P2X7R, microglia were seeded on coverslips in 12-well plates, treated with 250 nM WT or A53T α-Syn oligomer for increasing amounts of time (0–6 h), washed three times in PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 10% normal goat serum, the cells were incubated overnight at 4 °C with antibodies. After that, the cells were incubated with secondary antibodies conjugated to Alexa Fluor 488 or 546 (1:500, Molecular Probes, USA). Immunostaining was examined with a Nikon C2 + confocal microscope and photographed with a digital camera (CoolSNAP EZ, Photometrics).

Neurotoxicity assays

Resuspended exosomes (extracted from microglia from each group) were added to primary neuronal cultures for 24 h. Protein extraction and subsequent western blotting were performed as described above. The membranes were incubated overnight at 4 °C with primary antibodies against caspase-3. For morphological analysis of neuronal damage, MAP2 immunostaining was examined under a Nikon Eclipse TE2000E fluorescence microscope. All obtained images were imported into Image-ProPlus software version 7.0 (Media Cybernetics, Silver Spring, MD), to quantify the levels of MAP2 staining. The assessors were blinded to the experimental groups during image acquisition and quantification.

Statistical analysis

The data are expressed as the mean and standard deviation of at least three independent experiments. Analysis of the differences between group was performed by Student’s t-test. Significance was indicated by a p-value < 0.05. In all figures, the error bars represent the standard error of the mean. For all statistical analyses, measurements were taken from distinct samples.

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