Abstract Hippocampal neural stem/progenitor cells (NSPCs) are highly vulnerable to different stress stimuli, resulting in adult neurogenesis decline and eventual cognitive defects. Our previous study demonstrated that NOD-like receptor family pyrin domain-containing 6 (Nlrp6) highly expressed in NSPCs played a critical role in sustaining hippocampal neurogenesis to resist stress-induced depression, but the underlying mechnistms are still unclear. Here, we found that Nlrp6 depletion led to cognitive defects and hippocampal NSPC loss in mice. RNA-sequencing analysis of the primary NSPCs revealed that Nlrp6 deficiency altered gene expression profiles of mitochondrial energy generation and ferroptotic process. Upon siNlrp6 transfection, as well as corticosterone (CORT) exposure, downregulation of Nlrp6 suppressed retinoic acid-inducible gene I (RIG-1)/mitochondrial antiviral signaling proteins (MAVS)-mediated autophagy, but drove NSPC ferroptotic death. More interesting, short chain fatty acids (SCFAs) upregulated Nlrp6 expression and promoted RIG-1/MAVS-mediated mitophagy, preventing CORT-induced NSPC ferroptosis. Our study further demonstrates that Nlrp6 should be a sensor for RIG-1/MAVS-mediated mitophagy and play a critical role in maintain mitochondrial homeostasis of hippocampal NSPCs. These results suggests that Nlrp6 should be a potential drug target to combat neurodegenerative diseases relative with chronic stress. Keywords: NOD-Like receptor family pyrin domain-containing 6, Cognitive defects, NSPC ferroptosis, Mitochondrial disorders, Mitophagy Graphical abstract [39]Image 1 [40]Open in a new tab Highlights * • Nlrp6 depletion in mice causes hippocampal NSPC loss and cognitive defects. * • Nlrp6 deficiency altered gene expression profiles of energy generation and ferroptosis. * • Nlrp6 deficiency suppresses RIG-1/MAVS-mediated mitophagy that drives ferroptosis. * • SCFAs upregulate Nlrp6 to prevent CORT-induced NSPC ferroptosis. 1. Introduction Adult hippocampal neurogenesis participates in higher brain functions, but highly susceptible to multiple external factors include stress stimuli, aging and diet. The metabolism state of mitochondria controls the neurogenic capacity of hippocampal neural stem/progenitor cells (NSPCs) to modulate adult neurogenesis [[41][1], [42][2], [43][3], [44][4]]. For example, metabolic rewriting in glycolysis, oxidative phosphorylation (oxPhos), fatty acid biosynthesis and glutaminolysis mark the switch between cellular stages along the embryonic and adult neural stem cell lineage [[45][5], [46][6], [47][7], [48][8]]. However, accumulating evidence points to a role for stress-induced mitochondrial dysfunction in the decrease of NSPC proliferative capacity, which leads to the decline of hippocampal neurogenesis and the development of cognitive deficits [[49][9], [50][10], [51][11]]. It is well elucidated prolonged stress or glucocorticoid has a negative impact on mitochondrial genome and function, ultimately contributing to cell senescence and death [[52][12], [53][13], [54][14]], but little is known about how this happens. NOD-like receptor family pyrin domain-containing 6 (Nlrp6), a multifaced innate immune sensor, has been shown to initiate adaptive immune responses as a first line of defense in multiple tissues [[55][15], [56][16], [57][17]]. Distinct from the other NLR family members, Nlrp6 regulates inflammation in an inflammasome-dependent, as well as an inflammasome-independent pathway [[58]18]. It has been well documented that Nlrp6 in intestinal epithelial cells can sense the alterations of bacterial community and metabolites to regulates host defense during different microbial infections [[59][19], [60][20], [61][21]]. Nlrp6 regulates intestinal antiviral innate immunity by modulating retinoic acid-inducible gene I (RIG-1)/mitochondrial antiviral signaling proteins (MAVS) signaling pathway [[62]22]. More interesting, gastrointestinal Nlrp6 expression is inhibited by corticotropin-releasing hormone in mice with water-avoidance stress [[63]23]. In our recent study, Nlrp6 is highly expressed in hippocampal NSPCs, and down-regulated in response to chronic stress, as well as the major stress hormone, corticosterone [[64]24]. Knockout of Nlrp6 causes mitochondrial dysfunction and proliferation disorders in NSPCs, resulting in hippocampal neurogenesis decline in mice [[65]24]. This study strongly suggests that Nlrp6 may participate in the defense of NSPCs against external stimuli, however, the underlying mechanisms need to be further investigated. Emerging evidences suggest that iron-dependent ferroptosis plays a primary role in accelerating the processes of aging and neurodegeneration [[66][25], [67][26], [68][27]]. Ferroptosis is a non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation and metabolic constraints [[69]28]. Cell death in ferroptosis is mainly caused by the inactivation of cellular antioxidant system, especially the system xc^−/glutathione (GSH)/glutathione peroxidase 4 (GPx4)-dependent antioxidant defense [[70]29]. Herein, we found that Nlrp6 depletion in mice caused hippocampal NSPC loss and cognitive defects. RNA-sequencing data of the primary NSPCs were analyzed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA). It was revealed that Nlrp6 deficiency altered gene expression profiles of mitochondrial energy generation and ferroptotic process in Nlrp6-knockout (Nlrp6^KO) NSPCs. We also demonstrated that Nlrp6 deficiency caused a suppression of RIG-1/MAVS-mediated mitophagy that drove NSPC ferroptotic death by impairing the system xc^−/GSH/GPx4 axis. More interesting, short chain fatty acids (SCFAs) could upregulate Nlrp6 expression and promote RIG-1/MAVS-mediated mitophagy to prevent corticosterone-induced NSPC ferroptosis. Our study dropped a new hint for the resistant to hippocampal neurogenesis decline under chronic stress. 2. Materials and methods 2.1. Animal experiments C57BL/6 J male mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. According to the method of our previous study, a conventional knockout model of Nlrp6 (Nlrp6^KO mice) were constructed and purified [[71]24]. All animals were raised in Animal Research Center, School of Life Sciences, Nanjing University and treated in accordance with the relevant guidelines for animal experiments. The experimental personnel passed the test of experimental animal skills, and the experiment was approved by the Animal Ethics and Welfare Committee of Nanjing University (approval number: IACUC-2006017). At the age of 16 weeks, learning and memory function of WT and Nlrp6^KO mice was examined using Morris Water Maze (MWM) test and Novel object recognition (NOR) test. All behavioral tests were performed during an active period of animals’ light cycle (08:00–12:00). 2.1.1. MWM test MWM test was used to measure the hippocampus-dependent spatial learning. A white circular pool is artificially divided into four quadrants, and the wall of each quadrant has four patterns of different shapes and colors of red, yellow, blue and green as water entry points and spatial markers for mice. A circular transparent resin platform is 30 cm from the barrel wall, which is the target quadrant. Set the quadrant where the platform is located on the supporting software system as the third quadrant, the farthest quadrant as the first quadrant, and the other two quadrants clockwise, the total experimental time is 60 s, the platform dwell time is 5 s, and the experiment stops after the mouse mounts the platform for 5 s. The escape latency period is the period from the moment the mouse enters the water to climb the platform and stay for 5 s. 2.1.2. NOR test The NOR test was used to measure cognitive memory. In a plastic box with dim light, object A, object B, and object C to be identified, where A and B are squares with sides 3 cm long volume, C is a cylinder of similar volume. A video camera mounted on the wall directly above the box was used to record the testing session for off-line analysis. Mice acclimatize to their environment and explore freely in an unplaced chamber for 10min. After adaptation, put 2 identical objects A and B fixed positions at 8.5 cm away from the left and right sides of the box wall at the front 1/3 of the box, allow the mouse to explore for 10 min, record the trajectory, time and number of times the mouse explores the object, and remove the mouse after the end. Mice rest for 1h and then perform follow-up testing. Replace the B object with a C object with a different color and shape, the position is unchanged, put the same position into the mouse, explore again, and record the data. 2.1.3. Brain section and hippocampus tissue collection After the behavior tests, 3 mice of each group were anesthetized and perfused by heart with 4 % paraformaldehyde (PFA). Mouse brains were isolated and fixed in 4 % PFA over-night, and then dehydrated gradient concentration of sucrose solution. Following that, each brain was embedded in optimal cutting temperature compound (4583, Sakura Finetek, Torrance, CA, USA). Frozen coronal sections (30 mm thick) containing the hippocampus were obtained by using a freezing microtome (CM3050S, Leica, Wetzlar, Germany) and mounted on adhesion microscope slides (188105, Citotest, Jiangsu, China) for immunofluorescence staining. After mice were anesthetized and executed, the whole brain was isolated quickly. And then, the medial hippocampus was cut off and stored at -80 °C refrigerator, separately. 2.1.4. Immunofluorescent staining of brain section Three animals from each group were anesthetized and transcardially perfused with ice-cold 4 % paraformaldehyde. The brains were excised, fixed in 4 % paraformaldehyde overnight, incubated in 20 % sucrose/PBS overnight at 4 °C, and incubated in 30 % sucrose/PBS overnight at 4 °C. Then, the brain samples were frozen in optimum cutting temperature compound (Sakura Finetek, USA) and coronally cut with a cryostat into 30-μm-thick sections. The frozen sections were blocked with 10 % fetal bovine serum in PBS for 1h at room temperature. The slides were incubated with primary antibodies including mouse anti-NeuN (ab104224, Abcam) and rabbit anti-GFAP (Z0334, Dako) overnight at 4 °C and then incubated for 30min at 37 °C with Alexa Fluor-conjugated secondary antibodies (Invitrogen). The sections were counter stained with 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Images were captured using a Leica TCS SP8 confocal microscope. 2.2. Cell culture and treatment 2.2.1. Primary NSPCs extraction and culture 12-day pregnant mice were anesthetized with chloral hydrate and disinfected with 75 % ethanol. The embryo was taken out by opening the abdominal cavity, and the fetal mouse hippocampus was isolated. Transfer the hippocampus to a 15 mL centrifuge tube and digest with accutase enzyme for 10 min at 37 °C. And then, complete medium was added to terminate digestion and centrifugated at 1000 rpm, 4 °C for 3 min. Carefully discard the supernatant, resuspend the cell pellet in mouse neural stem cell complete medium, gently pipette, and centrifuge at 1000 rpm, 4 °C for 3min. After discarding the supernatant, resuspend the cell pellet in mouse neural stem cell complete medium and pass through a 70 μm screen, perform cell counting and seeding into a dish. Add 2–3 mL of fresh medium to the Petri dish on days 3 and 5 after cell inoculation, and primary NSPCs are collected after 7-day continuous culture. 2.2.2. NE-4c cell culture NE-4c cells (ZQ0275, ScienCell, Shanghai, China) were cultured in mouse NSC complete medium (J10001-4, ScienCell). The cells were seeded at 1.2 × 10^5 cells/mL density into poly-d-lysine-coated 25 or 75 cm^2 flasks. The medium was changed once a day. When the cells reached 90 % confluence, they were harvested with trypsinization in 37 °C cell incubator for 1min and then seeded in 6-, 24- or 96-well plates at 1.2 × 10^5 cells/mL density. 2.2.3. Cell treatments To alter the expression of Nlrp6 in NE-4c cells, plasmid pET-32 a-Nlrp6 or small interfering RNA of Nlrp6 as well as Lipofectamine 2000 (11,668,019, Invitrogen) transfection reagent were diluted in Opti-MEM medium (31,985,070, Gibco, Invitrogen) separately and left at room temperature for 5 min before they were mixed together. The plasmid mixed with lipofectamine 2000 was incubated at room temperature for another 20 min. Then mixture was added to the wells of culture plate containing Opti-MEM medium. The cells were incubated for 4 h, and then continued to be cultured in NSC complete medium. In addition, NE-4c cells were induced by 1 μm corticosterone (HY–B1618, MCE) for 48 h, or simultaneously treated by SCFAs, a mixture of 30 μm sodium acetate (S2889, Procell), 2 μm sodium propionate (P1880, Sigma-Aldrich), 1 μm sodium butyrate (820236, Sigma-Aldrich). According to experimental requirements, cells were cultured with 10 μm FCCP (HY-100410, MCE), an uncoupler of oxidative phosphorylation (OXPHOS) in mitochondria, for 2 h to induce mitophagy, 1 μm ferrostatin-1 (SML0583, Sigma-Aldrich) and 10 μm deferoxamine (HY–B1625, MCE) for 24 h to inhibit ferroptosis or 1 μm Poly (ic) (GC14710, GlpBio) for 24 h to activate RIG-1/MAVS axis. 2.3. Transmission electron microscopy (TEM) After 7-day culturation, primary NSPCs were washed with PBS (PH 7.4) and primarily fixed in 2.5 % glutaraldehyde for 24 h. The samples were post-fixed in 1 % OsO[4] for 1 h at RT, then progressively dehydrated through graded ethanol/acetone solutions and embedded in epoxy resin. Finally, ultrathin sections (70 nm) were prepared and stained with 2 % uranyl acetate followed by lead citrate. The images of cell ultrastructure were captured with a transmission electron microscope (HT7800, Hitachi, Tokyo, Japan). The experiments were repeated for three biological replicates. 2.4. Mito-tracker red staining Cultured cells were washed with PBS for 3 times and fixed by 4 % paraformaldehyde for 30 min. And then, cells were incubated with a QuickBlock™ Blocking Buffer with 0.1 % Triton™ X-100 for 1 h. After washing three times with PBS, cells were stained using Mito-Tracker Red CMXRos kit (C1049, Beyotime). Images were captured using a Leica TCS SP8 confocal microscope. 2.5. Cell survival assay Cell survival was measured by a CCK-8 kit (C0039, Beyotime). NE-4c cells with different treatments were seeded on the 96-well plate and then 1/10 volume of CCK-8 working solution (CCK-8 and culture medium 1:10 v/v) was added to each well. After 2 h of incubation at 37 °C, the optical density value at 450 nm was measured with a microplate reader (CLARIOstar, BMG Labtech, Germany). 2.6. MDA and GSH assay Cells were spread to six-well plates at a density of 1.2 × 10^5 cells/mL and collected after 48h of different treatments described above. The contents of MDA and GSH of cellular lysate were detected using the kits of MDA assay and GSH assay purchased from Beyotime Biotechnology (Shanghai, China). Protein concentration was detected using a bicinchoninic acid protein assay kit (23005, Thermo Scientific, Waltham, MA, USA) with bovine serum albumin as a standard. 2.7. Flow cytometry Cells were collected in 1.5 mL centrifuge tubes per 1 million, PBS washed, centrifuged and discarded supernatant, and the cells were treated using Autophagy Staining Assay Kit with MDC and PI (C3019S, Beyotime) according to the method described in the instructions. After assay, excess dye was washed off with PBS, and fluorescence detection was performed by flow cytometry (ThermoFisher Scientific). Cells were collected in 1.5 mL centrifuge tubes per 1 million, and were treated with a 5 μm DCFH-DA probe for 20 min, washed in PBS, and intracellular ROS levels were detected using flow cytometry. 2.8. RNA sequence (RNA-seq) data analysis The RNA of hippocampus and primary NSPCs used for RNA-seq was isolated with miRNeasy Micro Kit (1,071,023, Qiagen, Hilden, Germany) according to the manufacturer's protocol. The Illumina Novaseq platform was used to obtain multiplexed libraries, and then used the DESeq2 R package (1.16.1) to performed differential expression analysis, and the threshold of DESeq2 padj<0.05 and |log2FoldChange|≥2 were set for significantly differential expression. GO enrichment analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes (DEGs) using the David (Database for Annotation, Visualization, and Integrated Discovery) online tool. GSEA was performed to investigate gene expression changes in predefined sets of genes using GSEA v4.3.2 software. Mitochondria-related gene sets were obtained from MSigDB (Gene Set Resources for GSEA Software) and MitoCarta3.0 ([72]https://www.broadinstitute.org). Enrichment results for all gene sets obtained by RNA sequencing are represented by normalized enrichment scores (NES) and p-value values. 2.9. Western blotting Cells were harvested and homogenized in ice-cold RIPA buffer containing protease and phosphatase inhibitors. The lysates were obtained by centrifugation at 12,000 rpm at 4 °C for 10 min using a centrifuge (D1008, DLAB Scientific). Protein concentration was detected using a bicinchoninic acid protein assay kit (E112-02,Vazyme) with bovine serum albumin as a standard. Equal amount of protein of each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (IPVH00010, Millipore). Subsequently, the membranes were incubated in 5 % skimmed milk, probed overnight at 4 °C with primary antibodies and then incubated with HRP-conjugated secondary antibodies. Primary antibodies included rabbit anti-GPX4, rabbit anti-FTH1, rabbit anti-SLC7A11 (29650T, CST), mouse anti-Nlrp6 (A181108PA, Sigma-Aldrich), rabbit anti-NIX (CM-M139, Procell), rabbit anti-BNIP3 ([73]PB180322, Procell), mouse anti-RIG-1/DDX58 (67556, Proteintech), rabbit anti-MAVS (14341, Proteintech), rabbit anti-Tomm20 (00111633, Proteintech), mouse anti-LC3 (4108S, CST), rabbit anti-PINK1 (P0076, Sigma-Aldrich), mouse anti-β-actin (4970, CST), mouse anti-Tubulin (ab7291, Abcam) and mouse anti-GAPDH (ABS830030SS, ABSIN). The protein bands were detected by Tanon-5200 Chemiluminescence Imager (Tanon Science & Technology Co., Ltd., Shanghai, China). The density of bands was quantified using ImageJ (Version 1.50b, National Institutes of Health, USA), normalized to internal reference protein and expressed as fold change relative to the control value. 2.10. Real-time quantitative PCR (real-time qPCR) Total RNA was isolated using Trizol (10,596,026, Invitrogen), and was reversed into cDNA using qPCR Master Mix (Q311-02,Vazyme). Oligonucleotide primers ([74]Table 1) were purchased from Sangon Biotech (Nanjin, China). qRT-PCR was performed as previously described. Table 1. The primer sequences used in real-time qPCR analysis. Gene name Forward primers(5′-3′) Reverse primers(5′-3′) Mouse DDX58 AGAACAAACCGGGCAAC CATCAGCGACCGAGGTA Mouse MAVs TCTCTTGTCCATCTCAGTCCA TTCCCGATGTGCCTGTAGGA Mouse Nlrp6 CTGGCGTCATTGTGGAACCTCT TCTCACTCAGCTCCACAGAGGT Mouse SLC7A11 TGGGTGGAACTGCTCGTAAT AGGATGTAGCGTCCAAATGC Mouse FTH-1 GCCGAGAAACTGATGAAGCTGC GCACACTCCATTGCATTCAGCC Mouse GPx4 GTTTCGTGTGCATCGTCACC TATCGGGCATGCAGATCGAC Mouse Gclc ACACCTGGATGATGCCAACGAG CCTCCATTGGTCGGAACTCTAC Mouse Gclm TCCTGCTGTGTGATGCCACCAG GCTTCCTGGAAACTTGCCTCAG Mouse GSS CCAGGAAGTTGCTGTGGTGTAC GCTGTATGGCAATGTCTGGACAC Mouse GSR TGGCACTTGCGTGAATGTTG CGAATGTTGCATAGCCGTGG Mouse GAD1 CTGGCGTCATTGTGGAACCTCT TCTCACTCAGCTCCACAGAGGT Mouse GAD2 CCTTGCAGTGTTCAGCTCTCCT GCCTTGTCTCCTGTGTCATAGG Mouse SLC3A2 GCAGGATTAGAGCTGCCTCA CACCAGACCGTTCTTCTCCC [75]Open in a new tab 2.11. Luciferase reporter assay The 1 kb promoter of Nlrp6 was amplified using PCR with the following primer sequences: Forward,5′-CGGGGTACCGGCGACAGAACGAGAC-3’; Reverse, 5′-CCCAAGCTTGGGGTCTCTTCCTCC-3’. After purification, the PCR products were digested using KpnI/Hind III restriction enzyme and cloned into a pGL4.20 polyclonal vector. The luciferase reporter vector was transfected into NE-4c cells using Lipofectamine™2000 (11,668,030, ThermoFisher), and the relative luciferase activity in NE-4c cells was analyzed by Luciferase Assay System (E1501, Promega, Madison, WI, USA) 48 h after transfection. 2.12. Statistical analysis Statistical analyses were performed with GraphPad Prism Software 8.0 (GraphPad Software, San Diego, USA). Differences were analyzed by a two-tailed unpaired t-test or one-way analysis of variance (ANOVA) with post hoc tests for multiple group comparisons. P values < 0.05 were considered to indicate significance. 3. Result 3.1. Depletion of Nlrp6 causes hippocampal NSPC loss and cognitive defects in mice To investigate the role of Nlrp6 in the behaviors of spatial learning and memory, MWM and NOR experiments were performed in WT and Nlrp6^KO mice. Compared with WT mice, Nlrp6^KO mice took more time and total distance to find the platform in MWM test, but there was no statistical difference in the velocity during the training days ([76]Fig. 1A). Nlrp6^KO mice also spent less time and frequency in the target zone on the experimental day ([77]Fig. 1B). Moreover, the time and frequency exploring new objects in NOR test of Nlrp6^KO mice were less than that of WT mice ([78]Fig. 1C). Collectedly, Nlrp6 depletion caused the defects in the behaviors of spatial learning and memory in mice. Fig. 1. [79]Fig. 1 [80]Open in a new tab Nlrp6 depletion in mice caused hippocampal NSPC loss and cognitive defects. Learning and memory function of WT and Nlrp6^KO mice was examined at 16-week age. (A) Representative trajectory map and quantitative assessment of total distance and escape latency of the mice to the platform in MWM test (n = 7). (B) Representative trajectory map and quantitative assessment of crossing numbers and time spent of mice in the target quadrant in MWM test (n = 7). (C) Quantitative assessment of the number and time of the mice exporting old and novel objects in NOR test (n = 11–12). (D) GO enrichment analysis of hippocampal DEGs of Nlrp6^KOvs WT mice. (E) Representative images of Nestin and GFAP staining and quantitative assessment of positive cells in mouse hippocampal DG (n = 4). Scale bar, 200 μm. Data are expressed as mean ± SEM. Significance was calculated using a two-tailed unpaired t-test. *P < 0.05 and **P < 0.01 compared to WT group. RNA-seq analysis of hippocampal tissue identified a total of 596 genes that were differentially expressed by more than two-fold between Nlrp6^KO and WT mice. GO analysis reveals that most of these genes were enriched in brain functions related to learning, memory, nerve development, neuron differentiation and generation ([81]Fig. 1D). Furthermore, we marked NSPCs with antibodies against Nestin and GFAP in hippocampal DG region of mice using fluorescence immunohistochemistry. The results showed that the number of NSPCs (Nestin/GFAP positive cells) in hippocampal DG of Nlrp6^KO mice was much less than that of WT mice ([82]Fig. 1E). These results suggest that Nlrp6 depletion causes the loss of hippocampal NSPCs, contributing to cognitive defects in mice. 3.2. Depletion of Nlrp6 impaired mitochondrial morphology and function in NSPCs Next, the primary NSPCs isolated from WT and Nlrp6^KO fetal mouse were cultured to neutrospheres in vitro, respectively. The diameter of neurospheres grown from primary NSPCs of Nlrp6^KO mice was notably smaller than that of WT mice ([83]Fig. 2A). There was abnormal aggregation of mitochondria in Nlrp6^KO NSPCs indicated by an increase in the number of red fluorescence-stained cells in MTR staining ([84]Fig. 2A). The level of mitochondrial membrane protein, Tom20, in Nlrp6^KO NSPCs was also much higher than that in WT NSPCs ([85]Fig. 2B). Moreover, the aberrant mitochondria (yellow arrows) showing reduction even disappearance of mitochondria cristae and rupture of mitochondrial outer membrane was confirmed in Nlrp6^KO NSPCs by TEM characterization ([86]Fig. 2C). Fig. 2. [87]Fig. 2 [88]Open in a new tab Nlrp6 depletion altered mitochondrial morphology and functional gene profiles in NSPCs. The primary NSPCs were isolated from WT and Nlrp6^KO fetal mouse, and cultured for 7 days to neutrospheres, respectively. (A) Representative images of mitochondria labeled with red (Mito-Tracker Red CMXRos, MTR) and blue (DAPI). Scale bar, 100 μm. (B) The protein expression of TOM20 in neutrospheres (n = 4). (C) Representative images of transmission electron microscopy. Yellow arrow indicated the mitochondrion. Scale bar, 1 μm. (D) GO enrichment analysis of DEGs from Nlrp6^KOvs WT NSPCs. (E) Venn diagram and heat map of intersecting genes crossing mitochondrial genes from the Mitocarta 3.0 database and DEGs. (F) Enrichment plots of GSEA analysis. Data are expressed as mean ± SEM. Significance was calculated using a two-tailed unpaired t-test. **P < 0.01 and ***P < 0.001 compared to WT group. To systematically identify Nlrp6-controlled genes, we performed RNA-seq-based transcriptomic analysis of the primary NSPCs from WT and Nlrp6^KO mice. Total 3602 DEGs were identified, which was significantly enriched in multiple processes of mitochondrial function and components by GO analysis ([89]Fig. 2D). The altered metabolic pathways were associated with amino acid metabolism, carbohydrate metabolism, energy metabolism, glycan biosynthesis and metabolism, lipid metabolism by KEGG analysis ([90]Suppl Fig. 1A). Subsequently, mouse mitochondrial genes retrieved from the Mitocarta 3.0 database (Broad Institute) were intersected with DEGs and 157 intersecting genes underwent significant changes (i.e., with a p value < 0.05), with 20.4 % being up-regulated and 79.6 % being down-regulated ([91]Fig. 2E). To explore the NSPC-related cellular signaling regulated by Nlrp6, GSEA was performed in the MSigDB and MitoCarta3.0 database. GSEA revealed that Nlrp6 expression significantly correlated with mitochondrial biological pathways including aerobic electron transport chain (ETC), tricarboxylic acid (TCA) cycle, mitochondrial translation, mitochondrion localization ([92]Fig. 2F). Nlrp6 depletion caused the downregulation of core genes in ETC (including Cox, Uqcr, Nduf) and TCA cycle (including Aco2, Mdh2, Cs, Idh, Ogdhl) in NSPCs ([93]Supp Figs. 1B and C). These results indicate that mitochondrial energy generation is constrained in NSPCs with Nlrp6 depletion. 3.3. Nlrp6 depletion promotes ferroptotic NSPC death through the system xc^−/GSH/GPx4 axis By KEGG analysis, the down-regulated DEGs of Nlrp6^KO NSPCs were enriched in the pathways of ferroptosis, such as fatty acid degradation, p53 signaling pathway, GSH metabolism and peroxisome biogenesis ([94]Fig. 3A). GSEA analysis further indicated that ferroptotic process was involved in the death of Nlrp6^KO NSPCs ([95]Fig. 3B). A hierarchical clustered heatmap identified a series of up-regulated genes (such as ALOX12, Acsl4) linked to ferroptosis and down-regulated genes (such as Slc7a11, FTH1, GPx4, Ncoa4, Slc3a2) associated with anti-ferroptosis and anti-oxidation in Nlrp6^KO NSPCs ([96]Fig. 3C). These altered genes were confirmed in NE-4c cells with siNlrp6 transfection ([97]Suppl Fig. 2A). Furthermore, siNlrp6 transfection markedly decreased the percentage of cell survival, but increased the contents of lipid peroxidation and ROS levels in NE-4c cells, which were abolished by the ferroptosis inhibitors, Fer-1 and DFO ([98]Fig. 3D–F). Consistent with the reduction of GSH content ([99]Fig. 3G), the decreased expression of GCL, GSS and GSR were confirmed in NE-4c cells with siNlrp6 transfection ([100]Suppl Fig. 2B). In addition, corticosterone also induced a decrease in cell survival, an increase in lipid peroxidation and ROS levels, as well as a reduction of GSH contents in NE-4c cells, which were alleviated by Fer-1 and DFO ([101]Suppl Figs. 2C–F). Fig. 3. [102]Fig. 3 [103]Open in a new tab Nlrp6 depletion promotes ferroptotic NSPC death through the system xc^−/GSH/GPx4 axis. RNA-seq analysis of the primary NSPCs isolated from WT and Nlrp6^KO fetal mouse. (A) KEGG analysis of down-regulated genes of Nlrp6^KO NSPCs. (B) Enrichment plots in ferroptotic-related pathways by GSEA analysis. (C) Heat map of the core genes involved in ferroptosis. NE-4c cells transfected by control (CTL), siNlrp6 or simultaneously treated by Fer-1 and DFO. (D) Cell survival (n = 8). (E–G) MDA contents, ROS indicence and GSH contents (n = 4). (H–J) The protein expression of FTH1, SLC7A11 and GPX4 (n = 3). Data are expressed as mean ± SEM. Significance was calculated using a one-way ANOVA followed by the Dunnett's post-hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to CTL group. GPx4 is currently recognized as a central repressor of ferroptosis, and its activity depends on GSH produced from the activation of system xc^−, a cystine-glutamate antiporter (Slc7a11/Slc3a2) [[104]30]. Here, siNlrp6 transfection and corticosterone caused a decrease of ferritin heavy chain 1 (FTH1), Slc7a11 and GPx4 protein expression, which were also reversed by the ferroptosis inhibitors ([105]Fig. 3H–J, [106]Suppl Figs. 2G–I). These results suggest that Nlrp6 deficiency drives NSPC ferroptosis through disturbing the system xc^−/GSH/GPx4 axis. 3.4. Suppression of mitophagy contributes to NSPC ferroptosis induced by Nlrp6 depletion Mitochondrial autophagy (also known as mitophagy) is a critical process for cells to clear and recycle damaged mitochondria, which was closely associated to the process of cell ferroptosis [[107]31]. Here, GSEA revealed that Nlrp6 expression was also positively correlated with mitophagy signaling pathway, but not other autophagy-related signaling pathway. The core genes, such as Gba, Optn, Atg4, Sqstm1, were markedly decreased in Nlrp6^KO NSPCs ([108]Fig. 4A and B). Consistently, the number of MDC ^+ cells were significantly decreased in Nlrp6^KO NSPCs ([109]Fig. 4C). While, the levels of key proteins of classical autophagy pathway, PINK1, BNIP3 and NIX, were not changed in Nlrp6^KO NSPCs ([110]Fig. 4D–F). Moreover, the abnormal aggregation of mitochondria was accompanied by a significant decrease in the ratio of LC3II/I proteins in Nlrp6^KO NSPCs ([111]Fig. 4G and H). Fig. 4. [112]Fig. 4 [113]Open in a new tab Suppression of mitophagy contributes to NSPC ferroptosis induced by Nlrp6 depletion. The primary NSPCs were isolated from WT and Nlrp6^KO fetal mouse, and cultured for 7 days to neutrospheres, respectively. (A) Enrichment plots in mitophagy-related pathway by GSEA analysis. (B) Heat map of the core genes involved in mitophagy. (C) Flow cytometric analysis for the percentage of MDC positive cells (n = 7). (D–F) The protein expression of PINK1, BNIP3 and NIX (n = 3–4). FCCP was used to activate mitophagy. (G) Representative images of mitochondria labeled with red (Mito-Tracker Red CMXRos, MTR) and blue (DAPI). Scale bar, 100 μm. (H) The protein expression and the ratio of LC3II/I (n = 4). NE-4c cells transfected with CTL, siNlrp6 or simultaneously induced by FCCP. (I) Cell survival (n = 8). (J) The percentage of MDC positive cells (n = 4). (K) The protein expression and the ratio of LC3II/I (n = 4). (L–N) MDA contents, ROS indicence and GSH contents (n = 4). Data are expressed as mean ± SEM. Significance was calculated using a two-tailed unpaired t-test or a one-way ANOVA followed by the Dunnett's post-hoc test. *P < 0.05 and ***P < 0.001 compared to WT group or CTL group. A mitochondrial uncoupler, FCCP, was used to further evaluate the role of Nlrp6 in mitophagy. We found that FCCP treatment significantly increased the size of neutrospheres and reduced mitochondria aggregation in Nlrp6^KO NSPCs ([114]Fig. 4G). Consistently, the decease of LC3II/I ratio in Nlrp6^KO NSPCs was reversed by FCCP ([115]Fig. 4H). Moreover, we confirmed that siNlrp6 transfection caused a decrease of cell survival and MDC ^+ cells, as well as the ratio of LC3II/I protein in NE-4c cells, which were alleviated by FCCP ([116]Fig. 4I–K). FCCP ameliorated the increased MDA and ROS, as well as the decreased GSH ([117]Fig. 4L-N). These results indicate that the depletion of Nlrp6 in NSPCs leads to a decrease in mitophagy, but independent of the PINK1 and BNIP3/NIX axes. 3.5. Nlrp6 regulates mitophagy through RIG-I/MAVS axis It has been revealed that MAVS is required for healthy mitochondrial function by triggering RIG-I-mediated autophagy [[118]32]. Notably, compared to WT NSPCs, a significant decrease in the mRNA and protein levels of RIG-I (DDX58) and MAVS were observed in Nlrp6^KO NSPCs ([119]Fig. 5A–D). Here, a RIG-I agonist, poly (ic) was used to test the role of RIG-I/MAVS axis in the regulation of mitophagy by Nlrp6. As expected, the decrease of RIG-I protein, as well as the LC3-II/I ratio, in siNlrp6-interfered NE-4c cells were revised by poly (ic) ([120]Fig. 5E and F). Accordingly, poly (ic) also restored the decrease of RIG-I protein and LC3-II/I ratio in NE-4c cells induced by corticosterone ([121]Fig. 5G and H). Moreover, Nlrp6 overexpression prevented the decreases of RIG-I and MAVS proteins, as well as LC3-II/I ratio of corticosterone-induced NE-4c cells ([122]Fig. 5I-L). Therefore, it is speculated that Nlrp6 regulates mitophagy through RIG-I/MAVS axis, which is suppressed under chronic stress. Fig. 5. [123]Fig. 5 [124]Open in a new tab Nlrp6 regulates mitophagy through RIG-I/MAVS axis. (A–D) The mRNA and protein levels of DDX58 (RIG-1) and MAVS in primary NSPCs from WT and Nlrp6^KO mice (n = 3–4). (E, F) The protein levels of RIG-1 and LC3II/I of NE-4c cells transfected by CTL, siNlrp6 or simultaneously treated by Ploy (ic) (n = 4). (G, H) The protein expression of RIG-1 and LC3II/I in NE-4c cells induced by CTL, CORT or simultaneously treated by Ploy (ic) (n = 3). (I–L) The protein levels of Nlrp6, RIG-1, MAVS and LC3II/I in NE-4c cells induced by CTL, CORT or simultaneously transfected with Nlrp6 plasmid (n = 3–4). Statistical analysis of the protein levels was adjusted to loading control. Data are expressed as mean ± SEM. Significance was calculated using a two-tailed unpaired t-test or a one-way ANOVA followed by the Dunnett's post-hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to WT group or CTL group. 3.6. SCFAs up-regulates Nlrp6 to protect NSPCs from corticosterone-induced ferroptosis We previously found that SCFAs can upregulate Nlrp6 expression to protect mitochondrial function from corticosterone-induced damage in neural stem cells. To further verify the regulatory effect of SCFAs on Nlrp6, we constructed an Nlrp6 promoter luciferase reporter system and transfected it into NE-4c cells. Luciferase report assay showed that SCFAs significantly enhanced luciferase activity, suggesting that SCFAs may promote the activation of transcription factors on the target promoter, thereby upregulating the expression of Nlrp6 ([125]Fig. 6A). Western blot result confirmed that SCFAs also upregulated Nlrp6 proteins in corticosterone-induced NE-4c cells ([126]Fig. 6B). Furthermore, the decease of cell survival, the increase of MDA and ROS levels and the reduction GSH levels in corticosterone-induced NE-4c cells were alleviated by SCFAs treatment ([127]Fig. 6C–F). Moreover, the decease of autophagic ratio in corticosterone-induced NE-4c cells was also reversed by SCFAs treatment ([128]Fig. 6G). SCFAs also prevented the decrease of RIG-1 and MAVS proteins and LC3II/I ratio in corticosterone-induced NE-4c cells ([129]Fig. 6H–J). These data indicates that SCFAs up-regulates Nlrp6 and promote RIG-1/MAVS-mediated mitophagy, which may contribute to the protection for NSPCs against corticosterone-induced ferroptosis. Fig. 6. [130]Fig. 6 [131]Open in a new tab SCFAs up-regulates Nlrp6 to protect NSPCs from corticosterone-induced ferroptosis. (A) Nlrp6 luciferase reporter system and the activity of luciferase in NE-4c cells (n = 3). NE-4c cells were induced by CTL, CORT or simultaneously treated with SCFAs. (B) The protein expression of Nlrp6 (n = 4). (C) Cell survival (n = 7). (D–F) MDA contents, ROS indicence and GSH contents (n = 4). (G) The percentage of MDC positive cells (n = 9). (H–J) The protein expression of RIG-1, MAVS and LC3II/I (n = 4). Statistical analysis of the protein levels was adjusted to loading control. Data are expressed as mean ± SEM. Significance was calculated using a two-tailed unpaired t-test or a one-way ANOVA followed by the Dunnett's post-hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to CTL group. 4. Discussion Increasing experimental evidence supports a role for mitochondrial dysfunctions in hippocampal neurogenesis decline and cognitive defects induced by chronic stress. Our previous study demonstrates that Nlrp6 is essential to sustain NSPC mitochondrial homeostasis and proliferative capacity, which assists to prevent hippocampal neurogenesis decline under chronic stress [[132]24]. Here, hippocampal NSPC loss was also associated with cognitive defects in Nlrp6^KO mice. Functional enrichment analysis of RNA-sequencing data from the primary NSPCs by GO, KEGG and GSEA revealed that Nlrp6 depletion disrupted the signals and pathways of mitochondrial energy generation and triggered ferroptotic process in NSPCs. We also demonstrated that Nlrp6 deficiency led to the suppression of RIG-I/MAVS-mediated mitophagy that drove NSPC ferroptotic death by impairing the system xc^−/GSH/GPx4 axis. Moreover, SCFAs upregulated Nlrp6 and alleviated corticosterone-induced ferroptosis in NSPCs by promoting RIG-I/MAVS-mediated mitophagy. This study suggests that Nlrp6 is essential to protect against stress-induced NSPC damage in hippocampal neurogenesis. Given the general idea that the cellular processes of mitochondrial respiration and glutaminolysis to supply sufficient energy are the central regulators in the NSPC proliferative capacity [[133]7,[134]8,[135]33]. Mitochondrial respiratory function provides most of the cell energetic demands through the production of adenosine triphosphate (ATP) by glycolysis, TCA cycle and oxPhos occurring in the inner mitochondrial membrane through ETC under steady-state conditions. Hence, mitochondrial dysfunction underlies cognitive defects as a result of neural stem cell depletion and impaired neurogenesis [[136][34], [137][35], [138][36]]. Moreover, the altered expression of the Slc25 family genes encoding mitochondrial carrier proteins has been considered a marker of mitochondrial dysfunction in brain of mice with social defeat [[139]37]. In fact, stress and corticosteroids had a main effect on mitochondrial function genes, particularly the altered expression of NADH dehydrogenases and ATP synthase 6 genes [[140]13]. In this study, mitochondrial dysfunction in NSPCs with Nlrp6 deficiency was associated with the suppressed metabolic pathways involved in fatty acid degradation, amine acid metabolism, p53 signaling pathway, GSH metabolism and peroxisome biogenesis. GSEA analysis further revealed that most of down-regulated genes of Nlrp6^KO NSPCs were significantly related with the biological processes of ETC and TCA cycle, suggesting that mitochondrial respiratory function is impaired by Nlrp6 depletion. Moreover, glutaminolysis is another metabolic pathway that converts glutamine to glutamate by GLS, and then fuels the TCA cycle with α-KG to in turn produce more ATP during NSPCs proliferation or other emergency challenge [[141]7,[142]33]. In our system, the suppressed TCA cycle activity seems to drive the transform of glutamate to GABA shunt pathway by up-regulating GLS2 and GAD1/2. These results further suggest that the decreased proliferative capacity of NSPCs with Nlrp6 deficiency is at least partly due to the energy constraint. Ferroptosis, characteristic by iron dyshomeostasis, lipid peroxidation and perturbed expression of cystine/glutamate antiporter, is closely associated with neural loss in Alzheimer's disease [[143]38,[144]39]. Multiple studies have established a role of ferroptosis in cognitive impairment, suggesting the therapeutic potential of ferroptosis inhibition [[145][40], [146][41], [147][42]]. An ex vivo experiment using rat hippocampal slice culture showed that glutamate-induced neuronal excitotoxic death can be blocked by the ferroptosis inhibitor ferrostatin-1 [[148]43]. Neuron-specific GPx4 depletion causes neurodegeneration in vivo and ex vivo, highlighting the importance of this pathway in neuronal cells [[149]44]. Moreover, enhanced defense by overexpress Gpx4 against ferroptosis ameliorates cognitive impairment and reduces neurodegeneration in 5xFAD mice [[150]42]. In our study, GSEA analysis showed a significant correlation between Nlrp6 deficiency and genes associated with ferroptosis in NSPCs, which were confirmed in siNlrp6-interfered NE-4c cells and consistent with the accumulation of lipid peroxidation and ROS. Furthermore, the reduction of total GSH, as well as the decreased expression of GCL, GSS and GSR, indicates GSH metabolic disorder in siNlrp6-interfered NE-4c cells. The reduction of FTH1, SLCA11 and GPx4 expression were also observed in siNlrp6-interfered NE-4c cells. And that, these changes of lipid peroxidation, ROS and GSH levels, as well as FTH1, SLCA11 and GPx4 proteins, were reversed by ferroptosis inhibitors in siNlrp6-interfered NE-4c cells. Moreover, ferroptosis inhibitors could almost restore the decreased cell survival, of NE-4c cells induced by siNlrp6. These results strongly suggest that Nlrp6 deficiency drives NSPC ferroptosis by disturbing the system xc^−/GSH/GPx4 axis, which may play a major role in neuronal loss and cognitive impairment in Nlrp6^KO mice. Additionally, mounting evidence has proved the potential involvement of mitochondria in ferroptosis, but there are still some controversies [[151][45], [152][46], [153][47]]. Supporting this possibility, ferroptosis is associated with dramatic function and morphological changes of mitochondria in multiple disease states, especial neurodegenerative disease [[154][45], [155][46], [156][47], [157][48]]. In Nlrp6^KO NSPCs, the changed genes of mitochondrial function supported the disturbed metabolic pathways, such as GSH metabolism, glutaminolysis and peroxisome biogenesis, which were also associated with the ferroptotic process by GSEA analysis. Glutamine has been identified as the inducers of ferroptosis, thus enzymes (GLS) involved in glutaminolysis are potential therapeutic targets for ferroptosis inhibition [[158]49]. As well, inhibition of glutaminolysis restores mitochondrial function in senescent stem cell [[159]50]. In our study, the overexpressed GLS2 further indicates that the over activation of glutaminolysis participates in ferroptotic cell death of Nlrp6^KO NSPCs. Most notably, Nlrp6 depletion caused a driver of ferroptosis accompanied by the reduction of mitophagy, while, FCCP promoted the mitophagy and suppressed the ferroptotic process. Typically, mitophagy contributes to neuronal cell viability and offers protection through various mechanisms, including the removal of damaged mitochondria, which are primary sources of ROS production [[160]51,[161]52]. These results essentially highlight the role of Nlrp6 in protecting from stress-induced NSPC ferroptosis by regulating mitophagy to clear damaged mitochondria. In addition to their conventional role of meeting the cell's energy requirements, mitochondria also actively regulate innate immune responses against infectious and sterile insults [[162]53]. MAVS acts as a mediator of cross-talk between the immune response and metabolic regulation [[163]54,[164]55]. In turn, activation of RIG-I-mediated antiviral signaling triggers mitophagy through MAVS to maintain mitochondrial homeostasis [[165]32,[166]54]. More interesting, recent study has demonstrated that lipotoxicity reduces RIG-1 gene expression and activity leading to impaired autophagy and liver cell death [[167]56]. Here, we found that Nlrp6 depletion caused the suppression of RIG-1/MAVS-mediated mitophagy, most likely contributing to the aggregation of damaged mitochondria in NSPCs. Poly(ic) as a RIG-1 agonist activated of RIG-1/MAVS axis and promoted mitophagy, thereby preventing ferroptotic process in NSPCs with Nlrp6 deficiency. Moreover, upregulation of Nlrp6 by SCFAs treatment could also promote RIG-1/MAVS-mediated autophagy and prevent corticosterone-induced ferroptosis in NE-4c cells. These results strongly suggest that Nlrp6 should be a potential target for NSPC ferroptosis by modulating RIG-1/MAVS-mediated mitophagy. In conclusion, we have demonstrated that Nlrp6 deficiency causes the aggregation of damaged mitochondria, but suppresses RIG-1/MAVS-mediated mitophagy, which drives ferroptotic death in hippocampal NSPCs and leads to cognitive defects in mice. Corticosterone-induced NSPC ferroptosis is restored by SCFAs through upregulating Nlrp6 expression and promoting RIG-1/MAVS-mediated mitophagy, providing more evidence for the potential benefits of SCFAs on depression and cognitive dysfunction [[168]57,[169]58]. The evidence from this study suggests that Nlrp6 should be a sensor for RIG-1/MAVS-mediated mitophagy, which play a critical role in maintain mitochondrial homeostasis of hippocampal NSPCs to combat chronic stress. Formatting of funding sources This study was funded by the research start-up funds for new teachers from Nanjing Normal University and Jiangsu Provincial Key Construction Laboratory (No. SuJiaoKe [2024] 3) of Najing Xiaozhuang University. This study was funded by the research start-up funds for new teachers from Nanjing Normal University and State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University. CRediT authorship contribution statement Jingyan Shen: Writing – original draft, Project administration, Methodology, Data curation, Conceptualization. Pengfei Xie: Writing – original draft, Project administration, Methodology, Data curation. Junhan Wang: Writing – original draft, Software, Project administration, Methodology, Formal analysis, Data curation. Fan Yang: Project administration, Methodology. Shengjie Li: Software, Resources, Methodology. Haitao Jiang: Supervision, Software, Methodology. Xuefeng Wu: Writing – review & editing, Supervision, Conceptualization. Feng Zhou: Supervision, Project administration, Conceptualization. Jianmei Li: Writing – review & editing, Supervision, Funding acquisition. Declaration of competing interest The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All authors read and approved the final manuscript. Footnotes ^Appendix A Supplementary data to this article can be found online at [170]https://doi.org/10.1016/j.redox.2024.103196. Contributor Information Feng Zhou, Email: zfibcas@163.com. Jianmei Li, Email: lijianmei@njnu.edu.cn. Appendix A. Supplementary data The following are the Supplementary data to this article. Multimedia component 1 [171]mmc1.doc^ (11.1MB, doc) Multimedia component 2 [172]mmc2.docx^ (1MB, docx) Data availability No data was used for the research described in the article. References