Animals and treatment

iPLA2β knockout (iPLA2β−/−) mice were generated by Shanghai Model Organisms Center, Inc. All animal experiments followed protocols approved by the Institutional Ethics Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College. The mice were kept at the animal facility of the Chinese Academy of Medical Sciences with a12-h light–dark cycle and had free access to water and food. In our experiments, we used female iPLA2β−/− and C57BL/6J mice, aged 2–24 months. The mice were divided into four groups (n = 20 per group): 2-month-old C57BL/6J, 24-month-old C57BL/6J, 2-month-old iPLA2β−/−, and 24-month-old iPLA2β−/−. For AAV injections, 22-month-old female C57BL/6J mice received stereotaxic injections into the prefrontal cortex with either iPLA2β AAV (1ul, 1 × 1013 V.G./mL) or control AAV (1ul, 1 × 1013 V.G./mL). The mice were further divided into two groups (n = 20 per group) based on the type of AAV injected: control AAV and iPLA2β-AAV. The injections were administered using the following stereotaxic coordinates: AP 2.7, ML + 1.8, DV 2.0; AP 2.7, ML -1.8, DV 2.0. AAV injections were administered 21 days prior to formal behavioral testing [28]. Behavioral test training sessions were conducted two days before the formal testing began. Samples were collected on the 7th day following the initiation of the formal behavioral experiment. Figure 8a depicts the experimental design timeline, including the key time points for the assays and manipulations. The same set of animals was used for both Western Blot and PCR analyses to ensure consistency and comparability of results. Animals used for histology, including immunohistochemistry and immunofluorescence staining, were randomly selected from the same cohort as those used for Western blot and PCR.

Primary neurons culture

Primary neurons were isolated from the cortex of C57BL/6J mice on day 17 following the method outlined by Liu et al. [29]. For the D-gal (Sigma-Aldrich)-induced senescence model, primary neurons were cultured with 20 mg/mL D-gal in the culture medium for 48 h; For lentivirus mediated iPLA2β overexpression, purified lentivirus virus (2 μl, 4 × 108 TU/ml) was added to the culture medium for transduction.

Behavioral proceduresMorris water maze

The Morris water maze test was performed according to an established method [30]. Specifically, the test used a black circular pool divided into four quadrants (NE, NW, SE, and SW) and filled with water to a depth of 30 cm. A 10 cm platform was submerged 2 cm below the water surface and placed in the NE quadrant. Talcum powder was added to change the color of the water to white, facilitating the capture of images of the black mice using a camera. The water temperature was maintained constant from 23 °C to 25 °C throughout the experiment. The mice were subjected to five consecutive training trials. On the sixth day, a probe trial was conducted wherein the platform was removed and the mice were allowed to swim for 60s. Behavioral observations were captured using a camera positioned above the circular pool, and data analysis was performed using ANY maze software (SANS Biotechnology Co., Ltd).

Novel object recognition test

Novel object recognition test was conducted following the methods outlined in a published paper [28]. Each mouse underwent a 5-minute period of free exploration in an open-field box measuring 48 × 48 cm before formal testing. During the initial phase of the test, a cube-shaped object was placed within the box, and the mouse was given a 5-minute period for free exploration. Subsequently, a novel spherical object was introduced, and the mouse was given another 5 min to freely explore both objects. The time spent by the mice to investigate each object was recorded.

Elevated plus maze

The elevated plus maze test was conducted following the protocol outlined in a published paper [31]. Each mouse was positioned towards one of the open arms in the central connecting area and allowed to explore freely for 5 min. The time spent and number of entries into the open arms were recorded.

Rotarod test

Each mouse underwent adaptation training (SANS Biotechnology Co., Ltd.) before the formal experiment. During the formal test, the rotational speed of the rod was initially set to 5 rpm for 15 s, followed by a gradual increase to 50 rpm over a period of 5 min. Each mouse performed three repeat trials per session, with a 5-minute interval between trials for rest, and the average latency time of the three trials was recorded.

Adeno-associated virus production

The Pla2g6 sequence was inserted into the pAAV2-CMV_bGl-MCS-EGFP-WPRE-pA vector (Taitool Bioscience, Shanghai, China) to construct pAAV2-CMV_bGl-Pla2g6-2xFlag-HA-WPRE-SV40pA. The AAV9-Pla2g6 virus was generated by transfecting the overexpression construct and helper plasmids into AAV-293 cells. The AAV9-Pla2g6 virus was produced and purified using a modified standard protocol [32]. AAV9-CMV_bGI-EGFP-WPRE-pA (Taitool Bioscience, Shanghai, China) was used as control.

ATP level measurement

ATP levels were assessed using an ATP assay kit (Beyotime, S0026B) according to the manufacturer’s instructions. Briefly, the tissue or cell lysates were centrifuged at 12,000 × g for 5 min, and the resulting supernatant was used for analysis. Protein concentration was determined using a BCA kit (CWBIO, CW0014S).

Histology, immunohistochemistry (IHC) and immunofluorescence staining

For brain paraffin sectioning, the mice were perfused with PBS. Brain specimens were fixed in 4% paraformaldehyde (PFA) for 48 h, embedded in paraffin blocks, and sectioned into 6 μm slices. The sections were subsequently stained with hematoxylin and eosin (H&E) for histological examination. For immunohistochemistry (IHC), sections were deparaffinized and hydrated, followed by treatment with 10 mM citrate buffer (pH 6.0) and heating to boiling for 30 min. Endogenous peroxidase activity was blocked by treating the sections with 3% H2O2 for 10 min at room temperature. Then, brain sections were incubated overnight at 4 °C with the following antibodies: mouse polyclonal iPLA2β (sc-376563, 1:100, Santa Cruz), mouse monoclonal Iba1 (#17198, 1:400, Cell Signaling), or rabbit polyclonal GFAP (ab7260, 1:500, Abcam). Afterward, brain sections were incubated with anti-mouse and anti-rabbit secondary antibodies for 1 h at room temperature. Visualization of the protein/antibody complexes was achieved using DAB. The mPFC regions in the IHC sections were evaluated in a blinded manner. Histological changes were graded according to a previously described method [33]. The Histology score were graded in a blinded manner using a scoring system with the following criteria: Grade 0: Normal Limits-Tissue appears normal under the study conditions, accounting for factors such as age, sex, and strain. Any changes observed are within the expected range and are not considered pathological; Grade 1: Very Mild-Minimal changes are observed, just exceeding normal variation but with negligible pathological significance; Grade 2: Mild-Clear pathological lesions are present but are limited in extent and do not impair tissue function; Grade 3: Moderate-Pathological lesions are prominent and widespread, affecting tissue integrity and function; Grade 4: Severe-Extensive and severe pathological lesions, compromising the entire brain structure and function.

For brain cryosections used for immunofluorescence staining, mice were perfused with PBS, followed by 4% PFA. The brains were dissected and fixed in 4% PFA for 48 h. Subsequently, the brains were sequentially dehydrated in 15%, 20%, and 30% sucrose solutions, followed by embedding in OCT compound and sectioning into 10 μm slices [34]. For cell samples used in immunofluorescence staining, fixation was performed using 4% PFA for 15 min. Both brain sections and cells were permeabilized with 0.1% Triton X-100 in PBS, followed by incubation with primary antibodies at 4 °C overnight. Subsequently, sections and cells were incubated with Alexa-conjugated secondary antibodies for 1 h at room temperature before visualization. DAPI was applied for nuclear counterstaining. The primary antibodies employed in this study included iPLA2β (sc-376563, 1:100, Santa Cruz), NeuN (ab177487, 1:200, abcam), Iba1 (#17198, 1:400, Cell Signaling), GFAP (ab7260, 1:500, Abcam) and TOM20 (A19403, 1:200, ABclonal). The mPFC regions and cells on IF slides were evaluated in a blinded manner.

TUNEL staining

An In Situ Cell Death Detection Kit (Cat. #11684809910; Roche Applied Science) was used in this study. The sections were permeabilized with 1% Triton X-100 and 0.1% sodium citrate, followed by rinsing and staining according to the manufacturer’s guidelines.

SA-β-gal staining

For animal experiments, the mice were perfused with PBS. The brains were dissected and embedded in OCT compound and sectioning into 20 μm slices. For fresh cell coverlids and brain slices, SA-β-gal staining was performed using a Senescence β-Galactosidase Staining Kit (#9860; CST). Briefly, brain sections or cells were fixed in a 1 × fixative solution for 15 min, followed by two rinses in 1X PBS. Brain sections or cells were immersed in Staining Solution and incubated at 37 °C. Subsequently, the cells were washed thrice with PBS, and the positive cells were observed under a microscope.

q-RT-PCR

Total RNAs was extracted using the Eastep® Super Total RNA Extraction Kit (cat#LS1040, Promega). Reverse transcription was performed using the GoScriptTM Reverse Transcription Mix kit (cat#A2800, Promega). q-RT-PCR was performed utilizing the GoTaq® qPCR Master Mix (cat#A6001, Promega) on a Bio-Rad CFX96 TOUCH instrument. q-RT-PCR was performed using the SYBR green method as we previously described [27]. The expression levels of the target genes were evaluated by the Δ–Ct method and normalized to Gapdh. The specific primers were used: mouse Gapdh (NM_001289726.2)-forward: 5′- TGTGTCCGTCGTGGATCTGA-3′, -reverse: 5′- TTGCTGTTGAAGTCGCAGGAG-3′; mouse iPLA2β/ Pla2g6 (NM_016915)-forward: 5′-AGATGTCTTTCGTCCCAGCA-3′, -reverse: 5′- ATGATGTCGGACCCTAGCTG-3′; mouse sPLA2/ Pla2g2a (NM_001082531)-forward: 5′-GGCTGTGTCAGTGCGATAAA-3′, -reverse: 5′- TTTGGGCTTCTTCCCTTTGC-3′; mouse iPLA2γ/ Pnpla8 (NM_026164)-forward: 5′-TCGGTGGTTACATTGGTGGA-3′, -reverse: 5′- GCTGAAGTAGCACACGCTTT-3′; mouse TGF-β (NM_011577)-forward: 5′- GGCTACCATGCCAACTTCTG-3′, -reverse: 5′- CGTAGTAGACGATGGGCAGT-3′; mouse TNF-α (NM_013693.3)-forward: 5′-CCAACGGCATGGATCTCAAA-3′, -reverse: 5′-CCCTTGAAGAGAACCTGGGA-3′; mouse IL-1β (NM_008361.4)-forward: 5′- ACTCATTGTGGCTGTGGAGA-3′, -reverse: 5′- AGCCTGTAGTGCAGTTGTCT-3′; mouse CCL2 (NM_011333.3)-forward: 5′- GCTGGAGAGCTACAAGAGGA-3′, -reverse: 5′- ACCTTAGGGCAGATGCAGTT-3′.

Western blot analyses

Cells were homogenized in RIPA buffer (CW2333, CWBIO) and gently pipetted or vortexed to ensure complete cell lysis. Mice were anesthetized, and their brains were extracted and placed in ice-cold PBS. Brains were dissected to isolate the prefrontal cortices. The prefrontal cortices were specifically dissected and homogenized in RIPA buffer (CW2333, CWBIO) with a tissue homogenizer (KZ-III-F, Sercevio). Homogenized samples were then incubated on ice for 30 min. Following centrifugation at 13,000 × g for 10 min, the supernatant was collected. Protein concentrations were determined using the BCA Protein Assay Kit (CW0014S, CWBIO) and protein concentrations were between 3 and 5 ug/ul. Approximately 20 μg of sample proteins underwent 8–12% SDS-PAGE gel electrophoresis and were subsequently transferred onto PVDF membranes (Bio-Rad, USA). After blocking with 5% non-fat milk solution, the blots were incubated overnight at 4 °C with primary antibodies and subsequently incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The primary antibodies employed in this study included iPLA2β (#sc-376563, 1:200, Santa Cruz), P62(#5114, 1:1000, Cell Signaling), P16(#29271, 1:1000, Cell Signaling), Optineurin (#70928, 1:1000, Cell Signaling), PINK(23274-1-AP, 1:500, Proteintech), Parkin (#2132, 1:1000, Cell Signaling), Tom40 (sc-365467,1:500, Santa Cruz), MFF (#84580, 1:1000, Cell Signaling), LC3B (#83506, 1:1000,Cell Signaling), BNINP3(#3769, 1:1000,Cell Signaling), Nix (#12396, 1:1000, Cell Signaling), and β-actin (#4967, 1:1000, Cell Signaling). The protein bands were visualized using an ultrasensitive ECL chemiluminescence detection kit (PK10003, Promega) and captured using a ChemiDocTM Touch Imaging System (Bio-Rad, USA). ImageJ software was used to analyze the density of the protein bands (WB raw blots are provided in Additional file 10).

Transmission electron microscopy

Fresh mouse frontal cortex tissue samples (1 mm × 1 mm × 1 mm) were placed in fixation solution (0.1 M Phosphate Buffer containing 2.5% glutaraldehyde and 2.0% paraformaldehyde) and fixed at 4 °C for 4 h. After being washed three times with 0.1 M phosphate buffered saline, the frontal cortex tissues were post-fixed with 1% osmium tetroxide for 2 h. The tissue was rinsed three times with 0.1 M Phosphate Buffer for 15 min each. Dehydration was carried out by sequential immersion in 50%, 70%, 80%, 90%, 95%, and 100% ethanol, followed by 100% acetone for 15 min each. The tissue was then infiltrated with a series of mixtures of acetone and Epon 812 resin and finally embedded in pure Epon 812 resin. Ultrathin sections of the embedded samples were obtained using an ultramicrotome. The sections were double-stained with uranyl acetate and lead citrate and air-dried overnight. Observations and image acquisition were performed using a transmission electron microscope (HITACHI HT7800, Japan).

GFP-LC3 and Mito-DsRed analyses

48 h after transfection with AAV9-Pla2g6 or AAV9-CMV_bGI-EGFP-WPRE-pA, the primary cultured neurons were incubated in neurobasal FBS containing 20 mg/ml D-gal for 48 h. Next, neurons were transfected with AAVs containing GFP-LC3B and Mito-DsRed and allowed to recover for 48 h in growth media.

LC-MS/MS analysis

Lipidomic analysis was performed using an established protocol [35] (Biotree, Shanghai, China). For lipid extraction, 10 mg of the brain sample was homogenized in 400 μL H2O. Subsequently, the extract solutions were added to the brain homogenates and mixed thoroughly. The mixtures were centrifuged, and the supernatants were collected. An equivalent amount of MTBE was added to the supernatant, which was sonicated and centrifuged to isolate the supernatant. The supernatant was evaporated under vacuum and reconstituted. LC-MS/MS analysis was performed using a UHPLC system (1290, Agilent Technologies) with an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm). Biobud-v2.0.7 Software was used for quantification.

Statistics

Statistical analyses were performed using GraphPad Prism version 8. Results are presented as the mean ± SEM. Normality of data distribution was assessed using the Shapiro–Wilk normality test. For normally distributed data, comparisons between two groups were performed using a two-tailed Student’s t-test with Welch’s correction (*p < 0.05, **p < 0.01, ***p < 0.001). For non-normally distributed data, comparisons between the two groups were made using the Mann-Whitney U test. For comparisons involving more than two groups with normally distributed data, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used. For non-normally distributed data involving more than two groups, the Kruskal-Wallis test followed by Dunn’s multiple comparison test was applied (*p < 0.05, **p < 0.01, ***p < 0.001).