Chuanfeng Tang,Qiaona Wang,Jingyan Shen,Congying Wang,Hong Ding,Shiyu Wen,Fan Yang,Ruiqing Jiao,Xingxin Wu,Jianmei Li,Lingdong Kong
Neuron stem cell NLRP6 sustains hippocampal neurogenesis to resist stress-induced depression
NLRP6,Neural stem cells,Stress resilience,Depressive-like behaviors,ECRG4,Mitochondrial function,Proliferation,Pioglitazone
Neurogenesis decline in hippocampal dentate gyrus (DG) participates in stress-induced depressive-like behaviors, but the underlying mechanism remains poorly understood. Here, we observed low-expression of NOD-like receptor family pyrin domain containing 6 (NLRP6) in hippocampus of stress-stimulated mice, being consistent with high corticosterone level. NLRP6 was found to be abundantly expressed in neural stem cells (NSCs) of DG. Both Nlrp6 knockout (Nlrp6−/−) and NSC-conditional Nlrp6 knockout (Nlrp6CKO) mice were susceptible to stress, being more likely to develop depressive-like behaviors. Interestingly, NLRP6 was required for NSC proliferation in sustaining hippocampal neurogenesis and reinforcing stress resilience during growing up. Nlrp6 deficiency promoted esophageal cancer-related gene 4 (ECRG4) expression and caused mitochondrial dysfunction. Corticosterone as a stress factor significantly down-regulated NLRP6 expression, damaged mitochondrial function and suppressed cell proliferation in NSCs, which were blocked by Nlrp6 overexpression. ECRG4 knockdown reversed corticosterone-induced NSC mitochondrial function and cell proliferation disorders. Pioglitazone, a well-known clinical drug, up-regulated NLRP6 expression to inhibit ECRG4 expression in its protection against corticosterone-induced NSC mitochondrial dysfunction and proliferation restriction. In conclusion, this study demonstrates that NLRP6 is essential to maintain mitochondrial homeostasis and proliferation in NSCs, and identifies NLRP6 as a promising therapeutic target for hippocampal neurogenesis decline linked to depression.
Neuron stem cell NLRP6 sustains hippocampal neurogenesis to resist stress-induced depression

Neurogenesis decline in hippocampal dentate gyrus (DG) participates in stress-induced depressive-like behaviors, but the underlying mechanism remains poorly understood. Here, we observed low-expression of NOD-like receptor family pyrin domain containing 6 (NLRP6) in hippocampus of stress-stimulated mice, being consistent with high corticosterone level. NLRP6 was found to be abundantly expressed in neural stem cells (NSCs) of DG. Both Nlrp6 knockout (Nlrp6−/−) and NSC-conditional Nlrp6 knockout (Nlrp6CKO) mice were susceptible to stress, being more likely to develop depressive-like behaviors. Interestingly, NLRP6 was required for NSC proliferation in sustaining hippocampal neurogenesis and reinforcing stress resilience during growing up. Nlrp6 deficiency promoted esophageal cancer-related gene 4 (ECRG4) expression and caused mitochondrial dysfunction. Corticosterone as a stress factor significantly down-regulated NLRP6 expression, damaged mitochondrial function and suppressed cell proliferation in NSCs, which were blocked by Nlrp6 overexpression. ECRG4 knockdown reversed corticosterone-induced NSC mitochondrial function and cell proliferation disorders. Pioglitazone, a well-known clinical drug, up-regulated NLRP6 expression to inhibit ECRG4 expression in its protection against corticosterone-induced NSC mitochondrial dysfunction and proliferation restriction. In conclusion, this study demonstrates that NLRP6 is essential to maintain mitochondrial homeostasis and proliferation in NSCs, and identifies NLRP6 as a promising therapeutic target for hippocampal neurogenesis decline linked to depression.

Neurogenesis in mammalian hippocampal dentate gyrus (DG) region is unusual capacity to generate newborn neurons throughout the life,. Recent study shows that hippocampal neurogenesis is necessary and sufficient for conferring stress resilience in animals. The self-renewal capacity of radial glia-like neural stem cells (NSCs), a resident population in hippocampal DG, is fundamental to maintain the neurogenesis in response to stress in lifelong4, 5, 6, 7. Reduction of hypothalamic NSC proliferation rate, promotion of NSC senescence, as well as acceleration of aging-related physiological decline are observed in 14-month-old mice with Hnscr deletion. Of note, hippocampal neurogenesis-deficient mice show depressive-like behaviors with the hypersecretion of corticosterone, a stress factor in the hypothalamic-pituitary-adrenal axis of rodents in response to moderate psychological stressor restraint. The number of hippocampal NSCs is decreased in chronic restraint stress (CRS), or chronic corticosterone-stimulated mice, in parallel to pathogenesis of depressive-like behaviors and memory defect. These observations indicate that NSC reduction, proliferative capacity decline and senescence may underlie the defect of hippocampal neurogenesis under stress susceptibility. Nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 6 (NLRP6) is highly expressed in kidney, liver, small and large intestines10, 11, 12, 13, 14, 15. NLRP6 null mice are more susceptible to dextran sodium sulfate-induced colonic inflammation. Our previous study showed that fructose feeding down-regulated colonic NLRP6 expression accompanied with hippocampal neuroinflammation and neuronal loss, which were restored by a selective peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone (a well-known clinical drug). High level of corticotropin-releasing hormone (a known mediator of stress in hypothalamic-pituitary-adrenal axis) is reported to be released during water-avoidance stress and inhibit NLRP6 expression in intestinal epithelium of mice, which are also attenuated by PPARγ agonist rosiglitazone. However, few studies report the function of NLRP6 on central nervous system. In fact, brain cellular activity depends on high energetic support provided by mitochondria. Mitochondrial integrity is crucial for cell differentiation and dendritogenesis of newborn neurons. Mitochondrial function impairment such as decreased mitochondrial membrane potential and ATP production may diminish hippocampal neurogenesis in mice. Glucocorticoid and stress can affect the hippocampal mitochondrial function in rodents. Hence, maintaining NSC mitochondrial function and proliferation may be essential to buffer stress response and improve depressive-like behaviors. Here, we investigated whether NLRP6 sustained mitochondrial function and underlay NSC proliferation in hippocampal neurogenesis. The role and mechanism by which NLRP6 resisted stress-induced depression were also determined.

C57BL/6J male mice were purchased from GemPharmatech Co., Ltd. (Jiangsu, China) and kept in a specific pathogen-free facility in Animal Research Center of Nanjing University. All animal handling follows Animals (Scientific Procedures) Act, 1986 and associated guidelines and EU Directive 2010/63/EU for animal experiments. Animals’ husbandry and experiment procedures were approved by the Animal Ethical and Welfare Committee of Nanjing University (Approval No.: IACUC-2006017, China).

Mice were acclimated to the environment for 2 consecutive weeks, and then mice were subjected to different mild stressors for 5 weeks. In brief, mice were subjected to 2 or 3 mild stressors a day, such as restraint, wet bedding, no bedding, loud noise, cage titling (45°), stroboscopic illumination, inversion light/dark cycle, food restriction or tail suspension. Control mice without CMS were free to move in home cages and handled only for behavioral tests.

MS is a powerful paradigm of early life stress in rodents that triggers depressive-like behaviors21, 22, 23. MS of pups from their dam was conducted daily between 1:00 and 4:00 P.M., starting at postnatal Day 2 (P2) and terminating at P15 (MS mice). First, the newborn mice were removed from the home cage and placed into a clean cage. After the 3 h separation, newborn mice were returned to their home cage immediately. Control mice were standard facility-reared offspring without undergoing separation from their mothers. After birth, none of the control and MS mice were sexed and gave up to keep handling of all animals to a minimum disturbance.

According to the Nlrp6 gene structure, exon 1–8 of the Nlrp6-03 (ENSMUST00000183845.7) transcript was selected as the knockout region (about 7 kb). This project used CRISPR/Cas9 technology to modify the Nlrp6 gene. The brief process was as follows: transcribing sgRNA in vitro, and microinjecting Cas9 and sgRNA into the fertilized eggs of C57BL/6J mice to obtain F0 generation mice. The correct F0 generation positive mice and C57BL/6J mice were verified by PCR and sequencing to obtain a stable F1 generation positive mouse model. Nlrp6 alleles were genotyped from skin biopsies using PCR. C57BL/6J WT mice contained the entire mouse genome as a negative control, and a blank control without template was set. The primers used for genotyping were as following. Nlrp6-KO forward primer: ACTTAGCCATGTGGAGGTGTGC; Nlrp6-KO reverse primer: CCAGATGGCAATAGTGATCCACC; Nlrp6-WT forward primer: GAACTCAGACTCAGTCCCTGGTG; Nlrp6-WT reverse primer: TCAGCTCCTCCAGTGTAGCCAT.

Mice were divided into four groups receiving vehicle (control group), pioglitazone (HY-P1645, MedChemExpress, Monmouth Junction, NJ, USA), CMS, as well as CMS + pioglitazone (n = 12), respectively. These CMS mice were subjected to different stressors for 5 weeks. 30 mg/kg pioglitazone was administered to the mice by gavage once a day for 5 weeks. Mice without CMS were free to move in home cages and handled for behavioral tests.

WT and Nlrp6−/− mice grow naturally to around 13–14 weeks of age. Then the mice were equally divided into 6 groups (n = 6): WT, Nlrp6−/−, WT + pioglitazone, Nlrp6−/− + pioglitazone, WT + fluoxetine, Nlrp6−/− + fluoxetine. 30 mg/kg pioglitazone or 10 mg/kg fluoxetine (Sigma–Aldrich, St. Louis, MO, USA) was administered to mice by gavage once a day for 3 weeks,. Behavioral tests were then performed on the mice.

Mice with loxp sites flanking the exons of Nlrp6 (Nlrp6loxp/loxp) and Nestin-Cre transgenic mice were purchased from Shanghai Research Center For Model Organisms (Shanghai, China). Nlrp6loxp/loxp mice were mated with Nestin-Cre mice to generate Nlrp6-conditional knockout in NSCs (Nlrp6CKO) mice. The primers used for genotyping were as following. Nestin-Cre primer (sequence 5′–3′): P1: TTGCTAAAGCGCTACATAGGA (WT); P2: GCCTTATTGTGGAAGGACTG (common); P3: CCTTCCTGAAGCAGTAGA GCA (Mutant). Loxp primer (sequence: 5′–3′): P1: CTGCTCCCCACAAGGAACTC (Forward); P2: GGTGTTCCAAGGCTTCACCT (Reverse).

Total mRNA was isolated from tissues or cells using Trizol reagent (#15596026, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription was performed in a final volume of 20 μL containing 1 μg RNA using the HiScript II select qRT supermix (R222-01, Vazyme, Jiangsu, China). qRT-PCR was performed using gene specific primer sets and SYBR Green (Q311-03, Vazyme) on a qRT-PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). All primers were designed by ourselves and synthesized in the Generay Biotech Co., Ltd. (Shanghai, China). The reaction conditions were 95 °C for 30 s, 40 cycles of 95 °C for 10 s, 56 °C for 30 s and 72 °C for 30 s. Relative gene expression was calculated using the ΔΔCt method with β-actin as a reference gene. Primer sequences were shown in Supporting Information Table S1.

Blood samples and hippocampal homogenate of mice were centrifuged at 1500 rpm for 15 min using a centrifuge (D1008, DLAB Scientific, Beijing, China), and supernatant was collected. Corticosterone concentration was measured using corticosterone enzyme linked immunosorbent assay (ELISA) kit (KGE009, R&D Systems, Minneapolis, Minnesota, USA).

Each mouse was separated into a single cage for 3 days and then acclimated to two drinking bottles with normal water or 1% sucrose solution for 2 days. The position of the bottles was interchanged every 24 h. For preference test, mice were deprived of water for 24 h and then re-provided with two bottles for ensuing 2 h (8:00–10:00 P.M.). The total weight of each bottle was measured before and after the test to get the weight of liquid consumed. The sucrose preference was calculated as the fraction of the sucrose solution compared to the total amount of consumed liquid.

Briefly, OFT was performed in an open field box (Length, width, and height: 25 cm × 25 cm × 40 cm) wherein mice were placed close to against one side of the box wall (same for all mice). The mouse activity area was divided into nine squares of equal areas. During 10 min, Supermaze software was used to record time spent and distance travelled in the arena perimeter and center, as well as total distance travelled. Then mice were removed from the open-field box and returned to their home cages. After each trial, the objects and boxes were cleaned with a 70% ethanol to eliminate odor cues.

During the swimming adaptation phase, the mice were placed in a swimming environment for 5 min one day before the formal FST. Formal experiment on the second day, mice were individually placed into glass cylinders (50 cm high × 20 cm internal diameter) filled with water (23–25 °C) to a depth of 15 cm. Test sessions were recorded by a video camera positioned directly front the cylinders. A well-trained observer analyzed these data for the duration of mouse floating, swimming, and struggling with Supermaze software during the 5 min test period.

Mice were acoustically and visually isolated. Mice were individually suspended by the end of their tail with adhesive tape (about 50 cm above the floor) in a sound-isolated room. Each mouse was partitioned for a period of 6 min to avoid interference. Finally, the immobile and struggling time was exported from computer by trained investigators who were unaware of the grouping situation in a blinded manner using Supermaze software.

Mouse brains were fixed in 4% paraformaldehyde (PFA) overnight, dehydrated gradient concentration of sucrose solution, and embedded in optimal cutting temperature compound (4583, Sakura Finetek, Torrance, CA, USA). Frozen coronal sections (30 μm 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). NSCs or neuroectodermal (NE-4c) cells were fixed with 4% PFA for 15 min. The slices or cells were incubated for 1 h with 0.1% Triton X-100 in QuickBlock Blocking Buffer (P0235, Beyotime, Jiangsu, China), incubated with primary antibodies overnight at 4 °C, and incubated for 1 h at 37 °C with Alexa Fluor-conjugated secondary antibodies (Invitrogen). Hoechst (C1018, Beyotime) was used for nuclear staining. The primary antibodies included rabbit anti-IBA1 (019-19741, Wako, Saitama, Japan, ionized calcium-binding adapter molecule 1, microglia marker), mouse anti-NEUN (ab104224; Abcam, Cambridge, MA, USA), rabbit anti-DCX (ab77450, Abcam), rat anti-GFAP (13-0300, Invitrogen, glial fibrillary acidic protein, astrocyte marker), rabbit anti-KI67 (ab15580, Abcam), mouse anti-NESTIN (ab11306, Abcam), rabbit anti-NESTIN (ab221660, Abcam), rabbit anti-oligodendrocyte lineage transcription factor 2 (OLIG2, ab109186, Abcam, oligodendrocyte marker), rabbit anti-translocase of the outer membrane 20 (TOM20, 11802-1-AP, Protientech, Hubei, China) and mouse anti-TOM20 (ab56783; Abcam). Alexa Fluor-conjugated secondary antibodies included goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 555 (A-21424, Invitrogen) and Alexa Fluor 647 (A-21424, Invitrogen), goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (A-11008, Invitrogen) and Alexa Fluor 555 (A-21428, Invitrogen), donkey anti-goat IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 594 (A-21209, Invitrogen). Images were captured using a Leica TCS SP8 confocal microscope.

BeyoClick™ Edu Cell Proliferation Kit with Alexa Fluor 488 (C0071L, Beyotime) and or Alexa Fluor 555 (C0075L, Beyotime) was used for Edu staining, following to the manufacturer's protocols, respectively. In brief, a total of 1 × 105 cells/mL were seeded in 24-well plates containing coverslips. The Edu working solution was added to the final concentration (10 μmol/L) and incubated at 37 °C in 5% CO2 incubator for 2 h. After washing with PBS and fixing with 4% PFA, the coverslips were incubated with 0.1% Triton X-100 and dyed with reaction solution. Finally, the cells were stained with hoechst. The images were collected with Leica TCS SP8 confocal microscope. In addition, mice were injected intraperitoneally with Edu at dose of 50 mg/kg once a day for 10 days. After the mice were sacrificed, the remaining steps refer to cell manipulation.

Primary cells including NSCs, neurons, astrocytes and microglia were isolated from hippocampus of mice, respectively,25, 26, 27, 28.

Primary NSCs were isolated from 12-day mouse embryos. After the meninges and blood vessels were stripped off, the hippocampus of the brain was cut into pieces, transferred to 15 mL-centrifuge tube, and digested with accutase (SCR005, Millipore, Billerica, MA, USA) for 10 min at 37 °C. NSCs medium (CM-M139, Procell, Wuhan, China) was added to stop digestion, then centrifuged at 1000 rpm for 3 min using a centrifuge (5180 R, Eppendorf, Hamburg, Germany). Supernatant was carefully removed and abandoned. Cell precipitate was resuspended in NSCs medium and passed through a 70 μm-mesh sieve (93070, SPL life sciences, Pocheon, Korea). Finally, the cells were plated (1 × 106 cells/mL) into the flask and cultured in NSCs medium. Primary NSCs were cultivated for 7 consecutive days, and the diameter of neurospheres was recorded every day using an optical microscope (Olympus-IX53, Tokyo, Japan).

Briefly, hippocampus tissues from 16-day mouse embryos were collected in DMEM (C11995500BT Gibco, Invitrogen) at 4 °C and incubated in 2 mg/mL papain (HY-P1645, MedChemExpress) and 0.1 mg/mL DNAse (7900, StemCell Technologies Inc., Vancouver, Canada) for 30 min at 37 °C. DMEM medium containing 10% fetal bovine serum (FBS) (10099-141C, Gibco) was used to stop the digestion, and then single cell suspension was collected by pipetting step by step. Single cell suspension was passed through a 70 μm-mesh sieve (93070, SPL life sciences) and centrifuged at 1500 rpm at 4 °C for 5 min using a centrifuge (5180 R, Eppendorf). The upper liquid was discarded after centrifugation, primary neurons (2.5 × 104 cells/mL) were seeded in 6-well plates coated with poly-d-lysine (A-003-E, Millipore) containing DMEM medium (C11995500BT, Gibco) with 10% FBS. After 4 h of cell seeding, medium was instead by neurbasal Medium (21103049, Gibco) with B-27 Serum-Free Supplement (17504044, Gibco) to cultivate primary neurons. Half of the medium was changed every 2 days.

Primary astrocytes were isolated from hippocampus of pups aged between 2 and 4 days, and cultured on poly-d-lysine-coated cell culture plates in DMEM/F-12 (1×) (#11330032, Gibco) medium containing 10% FBS and 1% penicillin–streptomycin (V900929, Sigma–Aldrich). The meninges and blood vessels were removed, and hippocampus sample was transferred to 15 mL-centrifuge tube and digested with 0.25% trypsin (25200114, Gibco) in 5% CO2 incubator for 20 min. Then the trypsin was aspirated, and sample was washed by DMEM/F-12 three times and blown away in DMEM-F12 as much as possible. After standing the centrifuge tube for 2 min, supernatant was removed to a new 15 mL-centrifuge tube. We repeated the above steps for about 3–5 times until the supernatant was clear. Then the supernatant was sucked and centrifuged at 1000 rpm for 5 min using a centrifuge (5180 R, Eppendorf), and cell precipitate was re-suspended and plated in DMEM-F12 medium. After about 7–9 days of cell culture, with medium changed every 3 days, cells were adhered firmly and stratified. The lower layer was astrocytes and the upper layer was microglia. Microglia was separated by a 37 °C shaker at 200 rpm for 12 h using a thermostatic shaker (THZ-103 B, Bluepard, Shanghai, China). Then medium was collected and centrifuged at 1000 rpm using a centrifuge (5180 R, Eppendorf) for 5 min to obtain the microglia precipitation, and remaining adherent cells were astrocytes.

Cells or tissues 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 (23005, Thermo Scientific, Waltham, MA, USA) 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-NLRP6, rabbit anti-NESTIN, rabbit anti-GFAP (Z0334, Dako, Denmark), rabbit anti-IBA1, mouse anti-NEUN, rabbit anti-ECRG4 (SAB4503353, Sigma–Aldrich), mouse anti-β-actin (#4970, CST, Massachusetts USA), and mouse anti-Tubulin (ab7291, Abcam). 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 1.50 b, National Institutes of Health, USA), normalized to internal reference protein and expressed as fold change relative to the control value.

RNAscope technology provided by Advanced Cell Diagnostics (ACD, Hayward, CA, USA) allows the detection and evaluation of coinciding RNA expression profiles in different tissues or cells in unprecedented quantitative detail using multicolor fluorescent in situ hybridization. In this study, brain samples of mice were extracted, flash frozen in liquid nitrogen, and embedded in optimal cutting temperature compound. Frozen sections (10 μm thick) containing the hippocampus or colon were obtained by using a freezing microtome (CM3050S, Leica) and mounted on adhesion microscope slides. Slides were processed for 1-color RNA fluorescence according to the ACD RNAscope standard fluorescent protocol for fresh frozen tissue (ACD user manual document numbers 322380 & 322360-USM).

The CCK-8 assay (C0039, Beyotime) was used for the analysis of cell viability. NE-4c cells with different treatments were seeded on the 96-well plate. 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).

The RNA of primary NSCs and hippocampus of mice used for RNA-seq was isolated with miRNeasy Micro Kit (1071023, Qiagen, Hilden, Germany) according to the manufacturer's protocol. The RNA-Seq of primary NSCs and hippocampus in mice was performed and processed with standard protocols at OE Biotech Co., Ltd. and Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China), respectively.

IEM of hippocampus in mice was processed with standard protocols at the Analysis Center of Servivebio (Wuhan, China). Fresh blocks from hippocampal tissue were cut and harvested quickly. The 1 mm3 tissue blocks were transferred into an EP tube with fresh IEM fixative (#G1124-100 mL, Servicebio, Wuhan, China) for further fixation (at 4 °C for preservation and transportation). Hippocampus tissue blocks were washed on the ice box with pre-cooled 0.1 mol/L PBS (pH 7.4) for 3 times (10 min each), and then dehydrated and resin permeated. At 4 °C, the sample was put into the pure resin for embedding. The capsules with resin and samples were moved into low temperature. The samples were polymerized for more than 48 h at −20 °C by UV polymerizer, then ultra-thin sectioning and immunolabeling of polymerized samples were performed. The mitochondrial images of the Nestin-labeled NSCs in hippocampus were taken as the target under TEM (HT7800, Hitachi, Japan).

On the 7th day after NSC neurospheres culture, NSC precipitation was collected at 1000 rpm for 3 min using a centrifuge (5180 R, Eppendorf). TEM for NSC samples was processed with standard protocols at the Analysis Center of Servivebio. The fresh TEM fixative (G1102, Servicebio) was added to the tube and let the precipitation re-suspended in the fixative, and then fixed at 4 °C. Cells were fixed with 1% OsO4 in 0.1 mol/L PBS for 2 h at room temperature and rinsed 3 times in 0.1 mol/L PBS for 15 min. The cells were dehydrated with alcohol at room temperature and then experienced the steps of polymerization, ultra-thin section and staining. The cuprum grids were observed under TEM (HT7800, Hitachi) to take images for analyzing mitochondrial morphology.

NE-4c cells (ZQ0275, ScienCell, Shanghai, China) were cultured in mouse NSC complete medium (J10001-4, ScienCell). The cells were seeded at 1 × 105 cells/mL density into poly-d-lysine-coated 25 or 75 cm2 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 1 min and then seeded in 6-, 24-, 96-well plates at 1 × 105 cells/mL density according to different experimental requirements. NE-4c cells were treated by corticosterone (50–1600 nmol/L, HY-B1618, MedChemExpress) at about 50% confluence, then continued to cultivate until harvest. In the transfection step, plasmid Nlrp6 with no special tag, Nlrp6 with enhanced green fluorescent protein and Flag tag (Nlrp6-EGFP-Flag), Ecrg4 with Flag tag (Ecrg4-Flag) or small interfering RNA of Ecrg4 (si-Ecrg4), small interfering RNA of Nlrp6 and lipofectamine 2000 reagent (11668019, Invitrogen) were diluted in Opti-MEM medium (31985070, 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 under 37 °C in 5% CO2 incubator (41668241, Thermo Scientific) for 4 h, and then continued to be cultured in NSC complete medium. In addition, PPARγ agonist pioglitazone was used at a concentration of 10 μmol/L for 48 h in NE-4c cells, details please refer to the previous study.

NE-4c cells in different experimental processes were seeded in a poly-d-lysine-coated XF96 microplate (101085-004, Seahorse Bioscience, Agilent Technologies, CA, USA) at a density of 15,000 cells per well and incubated at 37 °C in 5% CO2 incubator. Basal oxygen consumption rate (OCR), ATP production and basal respiration were measured in the XFe 96 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent Technologies) using Mitochondrial stress assay, respectively. For data normalization, the total protein quantity was detected in each well by Micro BCA™ Protein Assay kit (23235, Thermo Scientific).

itochondrial membrane potential was evaluated by JC-10 assay (#22204, AAT Bioquest, CA, USA). NE-4c cells (1 × 105 cells/mL) were seeded on the 96-well plate. On the day of the experiment before harvest, the JC-10 kit was returned to room temperature first. JC-10 working solution (JC-10 and assay buffer A 1:100 v/v) was added with 100 μL/well to 96-well plates. The cells were incubated at 37 °C and protected from light for 1 h. The fluorescent intensity was measured at Ex/Em = 490/525 nm (green) and 540/590 nm (red) with a fluorescence microplate reader (CLARIOstar, BMG Labtech). The ratio of green/red fluorescent intensity was used to determine the mitochondrial membrane potential. Moreover, NE-4c cells (1 × 105 cells/mL) were seeded on 35 mm-confocal cell culture dish (801001, NEST Biotechnology, Wuxi, Jiangsu, China). Before images captured, JC-10 working solution was added to confocal cell culture dish, and cells were incubated in a 37 °C, 5% CO2 incubator for 30 min, protected from light. Images were captured using a Zeiss LSM 880 confocal microscope (Carl Zeiss, Jena, Germany).

Specific mitochondrial probe (MitoTracker Deep Red FM, M22426, Invitrogen) was used to detect the mitochondrial number in NE-4c cells. MitoTracker dyeing procedure refer to user guide of MitoTracker Mitochondrion-Selective Probes.

The 1 kb promoter of Nlrp6 was amplified by PCR using the following primers: Forward, 5′-CGGGGTACCGGCGACAGAACGAGAC-3′; Reverse, 5′-CCCAAGCTTGGGGTCTCTTCCTCC-3′. After purification, the PCR product was cloned into the multiple cloning sites of pGL4.20 vector after digested with Kpn I/Hind III restriction enzymes. NE-4c cells were transfected with luciferase reporter vector using lipofectamine 2000 Reagent. The relative luciferase activity in NE-4c cells was analyzed 48 h post-transfection using a Luciferase Assay System (E1501, Promega, Madison, WI, USA).

Data quantification was performed blindly and presented as mean ± standard error of mean (SEM). Two-tailed Student's t test was used for two group comparisons. And data were analyzed statistically using one-way ANOVA for comparisons of three and more groups with single factor variance, or two-way ANOVA for comparisons of four or more groups with double factor variance in this study. Value of P < 0.05 was considered statistically significant.

CMS and CRS were used as the stressors to investigate whether stress altered NLRP6 expression. CMS and CRS caused an intense reduction of Nlrp6 mRNA expression in hippocampus of mice with depressive-like behaviors, decreased body weight, increased serum and hippocampus corticosterone levels, respectively (Fig. 1A–D; Supporting Information Fig. S1). Interestingly, hippocampal Nlrp6 mRNA expression was negatively correlated with serum and hippocampal corticosterone level in these animals (Fig. 1E and F). To explore whether Nlrp6 deficiency was related with the development of depressive-like behaviors, Nlrp6−/− mice were generated (Fig. 1G). Generally, during the growth period of 16-week-old, body weight, food and liquid intake, and basal metabolic rate of Nlrp6−/− mice were not different from that of WT mice (Supporting Information Fig. S2). Energy expenditure, carbon dioxide excretion (VCO2), oxygen uptake (VO2), and respiratory exchange ratio (RER = VCO2/VO2) in Nlrp6−/− mice had no significant change compared with that in WT mice, showing a normal state of growth in Nlrp6−/− mice (Supporting Information Fig. S2C–S2J). On the other hand, data from SPT, FST and TST showed that Nlrp6−/− mice didn't have any behavior abnormality at young stage (4- or 8-week-old), but spontaneously developed depressive-like behaviors at 16- and 24-week-old; and Nlrp6−/− mice didn't show dyskinesia in OFT (Fig. 1H–K; Supporting Information Figs. S3–S6). These data indicate that, during process of growth, Nlrp6−/− mice are more likely to develop depressive-like behaviors than WT mice. To explore the role of NLRP6 in stress resilience, during postnatal Weeks 1–2, newborn WT and Nlrp6−/− mice stimulated daily by a 3 h MS protocol (an early life stress factor) were used (Fig. 2A). We found that these MS mice showed low Nlrp6 mRNA expression in hippocampus, as well as high serum and hippocampal corticosterone concentration compared with WT mice (Fig. 2B–D). The body weight of MS-stimulated Nlrp6−/− mice was not different from that of normally growing Nlrp6−/− animals during experimental MS period at 2–14 days after birth (Supporting Information Fig. S7A), but was significantly decreased at 4- and 8-week-old (Fig. S7B and S7C). Compared with WT mice, Nlrp6−/− mice developed notable depressive-like behaviors in SPT, FST and TST under condition of MS stimulation at 4- or 8-week-old, without the movement disorder in OFT (Fig. 2E–H; Fig. S7D–S7H). These data indicate that Nlrp6 deficiency impairs stress resilience in early life and then drives stress-induced depression in mice. Whereas, Nlrp6+/− mice showed no any behavioral change at 8-, 16- and 24-week-old compared with WT mice (Figs. S4–S6). Of note, CRS induced more severe depressive behavioral changes in FST and TST in Nlrp6+/− mice compared with WT mice at 8-week-old (Supporting Information Fig. S8). The behavioral performance of CRS-stimulated Nlrp6+/− mice also reflected the ability of NLRP6 to sense stress. Thus, Nlrp6-deficient mice seem more susceptible to stress than WT mice during growing up.

Hippocampal neurogenesis that newborn neurons are continuously generated and migrated into the granule cell layer (GCL) of DG, makes sense to avoid mental disorders,. Dense infiltrates of newborn neurons (positive for doublecortin, DCX) were observed in the subgranular zone (SGZ) of hippocampal DG, with an age-dependent loss from infant to adult in WT and Nlrp6−/− mice (Fig. 3A and B). Noteworthy, the number of newborn neurons in Nlrp6−/− mice was significantly less than that in WT mice at 16- and 24-week-old, but not at the young stage (2-, 3-, 4-, and 8-week-old) (Fig. 3A and B). To determine whether Nlrp6 deficiency was responsible for the decrease of newborn neuron generation in SGZ, mice were injected with Edu to label proliferating cells. The number of Edu and DCX co-labeled cells in Nlrp6−/− mice was less than in WT mice at 16 and 24 weeks of age (Fig. 3C and D). The defect of hippocampal neurogenesis in Nlrp6−/− mice coincided with the occurrence of depressive-like behaviors mentioned above at 16- and 24-week-old. Additionally, the number of astrocytes, microglia and mature oligodendrocyte in hippocampal DG showed no significant difference between WT and Nlrp6−/− mice at 16-week-old (Supporting Information Fig. S9). Nlrp6−/− mice suffering from MS developed depressive-like behavior at young stage as described above. Consistently, MS experience induced a significant decrease of newborn neurons as well as Edu and DCX co-labeled proliferated cells in Nlrp6−/− mice at young stage (4- and 8-week-old) compared with WT mice (Fig. 3E–G), indicating that hippocampal neurogenesis in Nlrp6-deficient mice displays substantial vulnerability to stress. Taken together, these data demonstrate that NLRP6 is necessary for maintaining hippocampal neurogenesis to confer stress resilience.

Next, hippocampal NLRP6 protein expression and cell distribution were analyzed in mice. NLRP6 protein was hardly expressed in whole mouse hippocampus (Supporting Information Fig. S10A). Then NLRP6 protein expression was determined in different nerve cells including NSCs, neurons, astrocytes and microglia isolated from hippocampus of mice, respectively (Fig. S10B). NSCs showed abundant expression of NLRP6 protein among hippocampal nerve cells (Fig. 4A). In hippocampus of WT mice, the distribution of Nlrp6 mRNA expression was confirmed using Nlrp6 mRNA label by a novel RNA in RNAscope with a unique probe design strategy. Nlrp6 mRNA was positively distributed in SGZ of hippocampal DG (Fig. S10C), and co-localized with NSCs and microglia instead of astrocytes (Fig. 4B; Fig. S10D). As shown in Fig. 4A, NLRP6 protein was highly expressed in NSCs, and the proportion of NSCs positive for Nlrp6 mRNA in RNAscope experiment was about 87.83 ± 2.65% (mean ± SEM, Fig. 4B). However, there was no significant change in microglia number, pathological injury and inflammation in Nlrp6−/− mice compared with WT mice (Figs. S9, S10E–S10H). Therefore, we investigated whether or how Nlrp6 deletion impacted the state of NSCs in SGZ of hippocampus. The number of NSCs was also age-dependently decreased both in WT and Nlrp6−/− mice (Fig. 4C and D). But at age of 16, 24, and 40 weeks, it was noted that NSC amount in Nlrp6−/− mice was less than in WT mice (Fig. 4C and D). Additionally, the number of KI67/NESTIN/GFAP positive cells in SGZ of Nlrp6−/− mice was less than that of WT mice at 16-week-old (Fig. 4E and F), suggesting that Nlrp6 deficiency impairs the capability of continuous self-renewal of NSCs. Furthermore, the positive regulators of cell proliferation, Ccna2, Ccnb1, Ccng2 and cyclin-dependent kinase (Cdk) 1 at mRNA levels were significantly decreased in hippocampus of Nlrp6−/− mice compared with WT mice (Fig. 4G). In addition, primary NSCs were isolated from the hippocampus and cultured in a proliferative medium. Primary NSCs formed free-floating cell aggregates that are called “neurospheres” in vitro (Fig. S10B). These primary neurospheres formed by NSCs from Nlrp6−/− mice were smaller, and the proportion of KI67 positive cells in neurospheres was also obviously lower than those from WT mice (Fig. 4H and I; Supporting Information Fig. S11A). The cell viability of NSCs from Nlrp6-deficient mouse was significantly lower than that of WT group (Fig. 4K). Taken together, Nlrp6 deficiency causes a reduced proliferation of NSCs in vivo. To further clarify the effect of low Nlrp6 expression in NSCs on depressive-like behaviors and neurogenesis, Nlrp6CKO mice were generated (Fig. 5A). Data from SPT, FST and TST showed that Nlrp6CKO mice hadn't dyskinesia in OFT, but developed depressive-like behaviors at 16-week-old compared with Nlrp6loxp/loxp mice (Fig. 5B–E, Supporting Information Fig. S12A and S12B). Moreover, the number of newborn neurons in Nlrp6CKO mice was significantly less than that in Nlrp6loxp/loxp mice at 16-week-old (Fig. 5F and G). The number of NSCs in hippocampus of Nlrp6CKO mice was decreased than that of Nlrp6loxp/loxp mice (Fig. 5H and I). In Nlrp6CKO mice, proliferative NSCs were less than that in Nlrp6loxp/loxp mice (Fig. 5H and J). These results demonstrate that Nlrp6 deletion impairs the capability of continuous self-renewal of NSCs, which limits the generation of newborn neuron, leading to depressive-like behaviors in mice.

Differential gene expression analysis in RNA-seq revealed 2519 up-regulated and 1239 down-regulated genes in hippocampus NSCs in Nlrp6−/− mice compared with WT mice (Supporting Information Fig. S13A). Gene ontology analysis for these up-regulated genes exhibited the enrichment of biological processes manifested as the inhibition of cell proliferation, mitochondrial membrane potential, related to brain developing, learning, memory, and aging (Fig. 6A). Down-regulated genes also showed obvious enrichment of biological processes related to regulation of cell proliferation, cell growth, and cellular senescence, and adjustment of mitochondrial membrane, electron transfer activity, ATPase activity, energy homeostasis and mitochondrial membrane permeability (Fig. 6B). In addition, changes in transcripts between NSCs from WT and Nlrp6−/− mice shown in volcano maps were significantly related to genes of mitochondrial function (Fig. 6C). Therefore, we hypothesized that NLRP6 may play a key role in NSC proliferation by defending mitochondrial function. To confirm this hypothesis, first, IEM using gold labeling on brain sections was performed to mark NESTIN protein for mapping the location of NSCs. IEM images of hippocampus showed that the structure of mitochondria in NSCs from 16-week-old Nlrp6−/− mice was seriously destructed, presenting swelling, deformation and cristae disappearance (Fig. 6D). Similarly, TEM results of primary NSCs cultured in proliferative medium for 7 days, showed that Nlrp6 deficiency caused a severe mitochondrial swelling, mitochondrial cristae disappearance, and mitochondrial membrane blur (Fig. 6E). And the mRNA expression levels of ATPase subunits including Atp2b1, Atp5g1, Atp5g2 and Atp5j2 were significantly decreased, while, of membrane traffickers including ATP8a2 and ATP9a were significantly increased in primary NSCs from Nlrp6−/− mice compared with WT mice (Fig. 6F). In consistent with qRT-PCR results of the positive regulators of cell proliferation in hippocampus (Fig. 4G), the mRNA expression levels of Ccna2, Ccnb1 and Cdk 1 were significantly decreased in primary NSCs from Nlrp6−/− mice compared with WT mice (Fig. S13B). These data indicate that Nlrp6 deficiency causes mitochondrial dysfunction, possibly contributing to the decreased proliferative capability of NSCs in vivo and in vitro. Exposure of a stress factor corticosterone significantly down-regulated NLRP6 mRNA and protein levels (Fig. 7A and B), and suppressed cell proliferation characterized by a reduction of cell viability and Edu positive cells in NE-4c cells (Fig. 7C–E). Coinciding with mitochondrial dysfunction in NSCs from Nlrp6−/− mice, seahorse analysis showed a significant reduction of OCR, basal respiration, and ATP production in NE-4c cells exposed to corticosterone (Fig. 7F–H; Supporting Information Fig. S14A–S14C). In this cell model, mitochondrial membrane potential was also significantly decreased (Fig. 7I and J). Normal mitochondrial morphology can range from small globular compartments to complex filamentous networks by immunofluorescent staining of anti-TOM20 antibody,. Mitochondrial compartmental protein TOM20 showed a fragmented and aggregated form around the nuclei in NE-4c cells exposed to corticosterone (Fig. 7K). In addition, corticosterone failed to change mitochondrial number in NE-4c cells (Fig. 7L and M). These features of mitochondrial dysfunction were in line with mitochondrial damage of NSCs in Nlrp6−/− mice detected by electron microscope mentioned above (Fig. 6D and E). Moreover, transfection of exogenous Nlrp6 with EGFP tag reversed proliferation decline, and significantly increased OCR, basal respiration, ATP production and mitochondrial membrane potential, as well as prevented TOM20 aggregation in NE-4c cells exposed to corticosterone (Fig. 7C–M; Fig. S14D and S14E). These data demonstrate that NLRP6 is required for maintaining mitochondrial homeostasis and proliferation in NSCs.

Here, we explored the molecular mechanism by which NLRP6 improved mitochondrial function and enhanced NSC proliferation. The RNA-seq data were further analyzed. Of note, an increase of Ecrg4 expression was observed in hippocampus and primary NSCs from Nlrp6−/− mice with stress susceptibility (Supporting Information Fig. S15A and S15B). ECRG4 has been reported as a negative regulator of the proliferation in senescent NSCs, and a factor for inhibiting mitochondrial membrane permeability transition in human T-leukemia cells. qRT-PCR detection further confirmed the enhanced Ecrg4 mRNA expression in hippocampus and primary NSCs from Nlrp6−/− mice compared with WT mice (Fig. 8A and B). ECRG4 protein levels were significantly increased in NE-4c cells exposed to corticosterone (Fig. S15C). In NE-4c cells, we found that knockdown of ECRG4 expression alone by transfection of si-Ecrg4 promoted cell proliferation (Fig. S15D). Overexpression of NLRP6 reduced ECRG4 protein levels to a normal value compared with corticosterone-exposed group (Fig. 8C). Knockdown of ECRG4 didn't change NLRP6 protein levels in NE-4c cells exposed to corticosterone (Fig. S15E), but improved cell proliferative capability manifested by increased cell viability and the number of Edu positive cell (Fig. 8D–F; Fig. S15F). Moreover, knockdown of ECRG4 protein increased OCR, basal respiration and ATP production, enhanced mitochondrial membrane potential, and prevented TOM20 aggregation, but had no effect on mitochondrial number in NE-4c cells exposed to corticosterone (Fig. 8G–M; Fig. S15G). To further address whether NLRP6 regulating ECRG4 protected mitochondrial function and maintained NSC proliferation, ECRG4 and NLRP6 were overexpressed in NE-4c cells (Supporting Information Fig. S16A). The data show that overexpression of ECRG4 abolished NLRP6 mediated-improvement of proliferative capacity in corticosterone-exposed NE-4c cells (Fig. S16B–S16D). Overexpression of both NLRP6 and ECRG4 decreased OCR, basal respiration, ATP production, and mitochondrial membrane potential, and led to TOM20 aggregation compared with NLRP6 overexpression in corticosterone-exposed NE-4c cells (Fig. S16E–S16L). Therefore, these results demonstrate that NLRP6 maintains mitochondrial homeostasis and supports NSC proliferation by suppressing ECRG4 expression.

PPARγ agonists is reported to up-regulate NLRP6 expression in colon of mice,. Here, Nlrp6-luciferase (luci-Nlrp6) plasmid was constructed, and PPARγ agonist pioglitazone increased the luciferase activity of NLRP6 in NE-4c cells (Fig. 9A). T0070907, a PPARγ antagonist, attenuated pioglitazone-induced luciferase activity increase of NLRP6 (Fig. 9B). In addition, pioglitazone promoted NLRP6 protein level of mouse primary NSCs and microglia instead of neurons and astrocytes (Supporting Information Fig. S17A). Pioglitazone increased NLRP6 and decreased ECRG4 at protein level in corticosterone-exposed NE-4c cells (Fig. 9C; Fig. S17B). It significantly attenuated corticosterone-induced decrease of proliferative capacity in NE-4c cells (Fig. 9D and E). Moreover, pioglitazone increased OCR, basal respiration, ATP production, and mitochondrial membrane potential, as well as prevented TOM20 aggregation in this cell model (Fig. 9F–L; Fig. S17C). To further demonstrate the critical role of NLRP6 as well as PPARγ in the protective effect of pioglitazone on mitochondrial function and proliferation of NSCs exposed to corticosterone, T0070907 and small interfering RNA were used to inhibit the expression of PPARγ and NLRP6, respectively (Supporting Information Fig. S18A). T0070907 or small interfering RNA of Nlrp6 blocked pioglitazone-mediated improvement of mitochondrial function and proliferative capacity in NE-4c cells (Fig. S18B–S18L). Finally, pioglitazone was found to improve depressive-like behaviors in CMS mice, but not affect the behaviors in normal animals (Fig. 10A–D; Supporting Information Fig. S19A–S19D). Pioglitazone promoted Nlrp6 mRNA expression and inhibited Ecrg4 mRNA expression (Fig. 10E and F), as well as improved hippocampal neurogenesis and enhanced the ability of NSC proliferation in hippocampus of CMS mice (Fig. 10G–K). Moreover, pioglitazone didn't alleviate depressive-like behaviors in Nlrp6−/− mice at 16-week-old (Supporting Information Fig. S20A–S20D), further demonstrating that pioglitazone improved depressive-like behaviors possibly in a NLRP6-dependent manner. Antidepressant fluoxetine is reported to attenuate corticosterone-induced depressive-like behaviors in mice by both neurogenesis-dependent and neurogenesis independent mechanisms. We found that fluoxetine improved depressive-like behaviors in Nlrp6−/− mice at 16-week-old (Fig. S20A–S20D). In addition, a stress factor corticosterone-stimulated mice or 16-week-old Nlrp6−/− mice showed depressive-like behaviors, but it is interesting that corticosterone-derived Nlrp6−/− mice didn't show more severe depressive-like behaviors than Nlrp6−/− mice (Supporting Information Fig. S21A–S21D). Thus, corticosterone-induced depressive-like behaviors may be mediated mainly by NLRP6-related NSC proliferation and hippocampal neurogenesis in mice. Taken together, NLRP6 protected against harm of stress factor on NSC proliferation dependently by suppressing ECRG4-mediated mitochondrial dysfunction. PPARγ agonist pioglitazone attenuated stress-induced mitochondrial dysfunction and proliferative capacity decrease by up-regulating NLRP6 expression in NSCs.

Resilience is the process of the adaptive response to a stressor. Neurogenesis decline in hippocampal DG promotes stress-induced depressive-like behaviors, and NSCs in hippocampal DG have the ability to maintain the neurogenesis in response to stress in lifelong,,. Yet, how NSCs sustain hippocampal neurogenesis and stress resilience remains incompletely defined. Here we found that stressors CMS and CRS down-regulated hippocampal Nlrp6 mRNA expression in hippocampus, being consistent with the development of depression and hypercorticosteronemia in mice. A subthreshold CMS (3-week mild stress) is used to divide mice into high-susceptible and low-susceptible subpopulations according to the sucrose preference, immobile time in TST and floating time in FST. The former may be associated with hippocampal interleukin-4 signaling suppression in mice. CRS model (14-day restraint stress) is reported to increase immobile duration in FST and decrease sucrose preference ratio in SPT, but subthreshold CRS (3-day restraint stress) cannot affect the two phenotypes in mice. In this study, by generating Nlrp6 knockout mice and analyzing their response to stress, we found that Nlrp6-deficient mice experienced MS showed significant depressive-like behaviors compared with WT at 4- and 8-week-old, respectively. These results suggested that Nlrp6-deficient mice were more susceptible to stress-induced behavioral changes than WT mice. Of note, without MS stimulation, Nlrp6-deficient animals developed depressive-like behavioral changes later at 16- and 24-week-old, but not in early life (at age of 4 and 8 weeks) compared with WT. It has also been reported that Nlrp6 knockout mice show a more dramatic drop and lower ability of stress resilience in sciatic function index immediately upon surgery than control group after peripheral nerve injury. Thus, hippocampal NLRP6 may be a sensor playing a critical role in sustaining stress resilience. The hippocampal DG is the critical region for emotional regulation requiring continuous neurogenesis to generate newborn neurons,. Silencing newborn neurons of hippocampal DG in mice increases the susceptibility to restraint stress and chronic social defeat,. In this study, relative to WT mice, Nlrp6−/− mice at age of 16 and 24 weeks, and Nlrp6−/− mice at age of 4- and 8-weeks experienced MS, showed less number of newborn neurons in hippocampal DG (Fig. 3). The defect of hippocampal neurogenesis in Nlrp6-deficient mice may be responsible for the reduction of stress resilience, contributing to the occurrence of depressive-like behaviors. Therefore, Nlrp6 deficiency impaired the capability of hippocampal neurogenesis under the stress compared with WT mice. However, corticosterone didn't aggravate depressive-like behaviors in Nlrp6−/− mice at 16-week-old, indicating that NLRP6 downregulation-related hippocampal neurogenesis defect may be the main factor contributing to corticosterone-induced depression. NSCs in hippocampus maintain the capability of self-renewal to sustain lifelong neurogenesis to generate newborn neurons. Here, we demonstrated that NLRP6 was essential to sustain the capability of NSC proliferation. Of note, NSCs show fading morphology during aging, being consistent with the observations that the reactive-like morphology of reactive NSCs is induced by hippocampal seizures,. Our observations from the NSCs morphology showed that, in WT mice, the majority of NSCs remaining in hippocampal DG presented a small and simple morphology with little branching at the young stage (4- and 8-week-old), but gradually become a big and complex morphology with extensive branching in the process of growth from 16 to 40 weeks of age. However, in Nlrp6−/− mice, a different location of NSCs was positioned into GCL and kept away from the hilus in hippocampus especially at 16, 24 and 40 weeks of age (Fig. 4C). NSCs in Nlrp6−/− mice showed a big and complex morphology with extensive branching and multipolar extensions even at young stage (4-, 8-, and 16-week-old). The morphology of NSCs in Nlrp6−/− mice had the possibility of premature senescence compared with WT mice, being parallel to the RNA-seq results, indicating that, Nlrp6−/− compared to WT NSCs, up-regulated genes were predicted to mediate brain developing, and aging, and down-regulated genes also to cellular senescence. To further explore whether NLRP6 modulated NSC proliferation for hippocampal neurogenesis in depression, Nlrp6CKO mice were generated. We found that Nlrp6CKO mice exhibited hippocampal neurogenesis defect and depressive-like behaviors at 16-week-old. These results demonstrated that Nlrp6 deficiency damaged the capability of continuous self-renewal and even drove the premature senescence of NSCs in depression. However, whether or how NLRP6 affects NSCs aging need further study in the future. Here, we didn't further prove the effect of mitochondrial damage on NSC proliferation. In fact, mitochondria homeostasis is prominent for NSC proliferation and hippocampal neurogenesis,,. Impaired mitochondrial function alters hippocampal neurogenesis. It remains unclear whether NLRP6 employs a mechanism to protect mitochondrial function and proliferative capacity in NSCs. Furthermore, the glucocorticoid response to stress is increased in neurogenesis-deficient mice with depressive-like behaviors. Hypersecretion of corticosteroids in response to chronic stress impairs hippocampal neurogenesis by suppressing NSC proliferation and neuronal differentiation in rodents46, 47, 48. In this study, mitochondria from hippocampus NSCs of Nlrp6−/− mice or cultured primary Nlrp6−/− NSCs showed swelling, deformation and cristae disappearance. Moreover, we found that corticosterone suppressed NLRP6 expression, and caused mitochondrial dysfunction and proliferation decrease in NE-4c cells. More importantly, overexpression of NLRP6 ameliorated these aberrances, supporting its prevention of NSC proliferation and mitochondrial dysfunction. RNA-seq data analysis and qRT-PCR validation revealed the increased mRNA expression of Ecrg4 in hippocampus and primary NSCs from Nlrp6−/− mice. ECRG4 is a secreted inducer of cell senescence as well as cell proliferation, and its overexpression is observed in aged precursor cells of central nervous system in mice,. The results from double overexpression of NLRP6 and ECRG4 in corticosterone-exposed NE-4c cells clarified the effect of NLRP6 on mitochondrial function and proliferative capacity by regulation of ECRG4. These findings demonstrated that NLRP6 protected against stress impact on NSC mitochondrial function in hippocampus possibly by monitoring ECRG4. NLRP6 is reported to regulate the expression or modification of some transcription factors (nuclear factor kappa B, signal transducer and activator of transcription 3, SMAD family member 2/3, nuclear factor erythroid 2-related factor 2), which may affect ECRG4 expression. The mechanism by which NLRP6 inhibited the expression of ECRG4 may involve the transcriptional regulation, which is deserved further study50, 51, 52, 53. Moreover, PPARγ agonist pioglitazone as well as overexpression of NLRP6 down-regulated ECRG4 expression, to reverse cell proliferation decline and repair mitochondrial function in corticosterone-exposed NSCs. Additionally, PPARγ antagonist (T0070907) or small interfering RNA of Nlrp6 was found to block pioglitazone-mediated improvement of proliferative capacity and mitochondrial function in corticosterone-exposed NE-4c cells. This study and previous studies have shown that pioglitazone rescues depressive-like phenotypes in high-fat diet-fed or CMS-stimulated mouse models,. Pioglitazone enhanced NLRP6 expression by activating PPARγ, and then down-regulated ECRG4 expression in corticosterone-exposed NSCs, indicating that pioglitazone may regulate PPARγ/NLRP6/ECRG4 signaling pathway in its attenuation of stress-induced depression. Pioglitazone didn't alleviate depressive-like behaviors of Nlrp6−/− mice at 16-week-old. Thus, NLRP6 may participate in the alleviation of depressive-like behaviors by pioglitazone. Our findings show that pharmacological enhancement of NLRP6 is significant to protect mitochondrial function and proliferative capacity in NSCs. Fluoxetine improved depressive-like behaviors of Nlrp6−/− mice at 16-week-old, which should be supported by the activation of the excitability of newborn neurons in mice exposed to stress,. Therefore, the neurogenesis disorder related to the downregulation of NLRP6 expression may be treated with the combination of PPARγ agonist (pioglitazone) and antidepressant (fluoxetine). The defective colonic NLRP6 inflammasome might be associated with hippocampal neuroinflammation and neuronal loss. Generally, neuroinflammation is suggested as a key factor of depression56, 57, 58, 59. Here, we didn't find significant neuroinflammatory responses including pathological change and inflammatory infiltration in hippocampus of Nlrp6−/− mice relative to WT mice. Being similar to some studies,,,, we considered that neurogenesis may be the culprit behind depression. The depressive-like behaviors in Nlrp6−/− mice may be more closely related to hippocampal NSCs, fortunately, which were proved again in Nlrp6CKO mice. Microbiome disruption or intestinal damage could influence depression62, 63, 64, whereas, NLRP6 is reported to protect the gut from damage induced by fructose or dextran sulfate sodium,,. Whether intestinal Nlrp6 deficiency has an indirect interference on depressive-like behaviors requires more studies in the future.

NLRP6 in NSCs controls ECRG4 expression to sustain mitochondrial homeostasis and proliferation for hippocampal neurogenesis. Stress-induced reduction of NLRP6 expression drives hippocampal neurogenesis decline and causes depression. Upregulation of NLRP6 by pioglitazone prevents mice from stress-induced depression. Our research identifies NLRP6 as a promising therapeutic target for hippocampal neurogenesis decline to treat stress-induced depression.