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Autophagic death of neural stem cells mediates chronic stress-induced …

November 7th, 2022 1:53 am

. 2020 Mar;16(3):512-530. doi: 10.1080/15548627.2019.1630222. Epub 2019 Jun 24. Seonghee Jung 1 ,Seongwon Choe 1 ,Hanwoong Woo 1 ,Hyeonjeong Jeong 1 ,Hyun-Kyu An 1 ,Hyewon Moon 1 ,Hye Young Ryu 1 ,Bo Kyoung Yeo 1 ,Ye Won Lee 1 ,Hyosun Choi 2 ,Ji Young Mun 3 ,Woong Sun 4 ,Han Kyoung Choe 1 ,Eun-Kyoung Kim 1 5 ,Seong-Woon Yu 1 5

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Seonghee Junget al. Autophagy. 2020 Mar.

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Macroautophagy/autophagy is generally regarded as a cytoprotective mechanism, and it remains a matter of controversy whether autophagy can cause cell death in mammals. Here, we show that chronic restraint stress suppresses adult hippocampal neurogenesis in mice by inducing autophagic cell death (ACD) of hippocampal neural stem cells (NSCs). We generated NSC-specific, inducible Atg7 conditional knockout mice and found that they had an intact number of NSCs and neurogenesis level under chronic restraint stress and were resilient to stress- or corticosterone-induced cognitive and mood deficits. Corticosterone treatment of adult hippocampal NSC cultures induced ACD via SGK3 (serum/glucocorticoid regulated kinase 3) without signs of apoptosis. Our results demonstrate that ACD is biologically important in a mammalian system in vivo and would be an attractive target for therapeutic intervention for psychological stress-induced disorders.Abbreviations: AAV: adeno-associated virus; ACD: autophagic cell death; ACTB: actin, beta; Atg: autophagy-related; ASCL1/MASH1: achaete-scute family bHLH transcription factor 1; BafA1: bafilomycin A1; BrdU: Bromodeoxyuridine/5-bromo-2'-deoxyuridine; CASP3: caspase 3; cKO: conditional knockout; CLEM: correlative light and electron microscopy; CORT: corticosterone; CRS: chronic restraint stress; DAB: 3,3'-diaminobenzidine; DCX: doublecortin; DG: dentate gyrus; GC: glucocorticoid; GFAP: glial fibrillary acidic protein; HCN: hippocampal neural stem; i.p.: intraperitoneal; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MKI67/Ki67: antigen identified by monoclonal antibody Ki 67; MWM: Morris water maze; Nec-1: necrostatin-1; NES: nestin; NR3C1/GR: nuclear receptor subfamily 3, group C, member 1; NSC: neural stem cell; PCD: programmed cell death; PFA: paraformaldehyde; PX: Phox homology; PtdIns3P: phosphatidylinositol-3-phosphate; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; SGK: serum/glucocorticoid-regulated kinases; SGZ: subgranular zone; SOX2: SRY (sex determining region Y)-box 2; SQSTM1: sequestosome 1; STS: staurosporine; TAM: tamoxifen; Ulk1: unc-51 like kinase 1; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; VIM: vimentin; WT: wild type; ZFYVE1: zinc finger, FYVE domain containing 1; Z-VAD/Z-VAD-FMK: pan-caspase inhibitor.

Keywords: Atg7 knockout; autophagic cell death; corticosterone; hippocampal neurogenesis; serum/glucocorticoid regulated kinase 3; stress.

Figure 1.

Chronic restraint stress (CRS)-induced reduction

Figure 1.

Chronic restraint stress (CRS)-induced reduction in the number of adult hippocampal NSCs is

Chronic restraint stress (CRS)-induced reduction in the number of adult hippocampal NSCs is prevented by Atg7 deletion. (A) Scheme of mouse breeding and experimental time line for generation of tamoxifen (TAM)-inducible NSC-specific Atg7-NSC cKO mice. (B) Gene dosedependence of ATG7 immunofluorescence intensities in the DG of the hippocampus. Scale bar: 50 m. Solid and dotted circles indcate granule neurons and NSCs, respectively. The graph shows quantification of SOX2+ and ATG7+ cells (n =6). (c) Timeline of the experiment. (D) Representative images of BrdU and SOX2 staining in the subgranular zone (SGZ) of the hippocampus. Scale bar: 20 m. The graph on the right shows quantification of BrdU+ and SOX2+ cells (n =68 per group). (E) Immunofluorescence of SOX2, NES and VIM in the SGZ of the DG of the hippocampus. Scale bar: 50 m. Arrows indicate NSCs positive for each marker. ***P <0.001 for the total SOX2+ cells. ###P <0.001 for BrdU+ SOX2+ cells. n.s., not significant.

Figure 2.

CRS induces autophagy in adult

Figure 2.

CRS induces autophagy in adult hippocampal NSCs. ( A ) Work flow of

CRS induces autophagy in adult hippocampal NSCs. (A) Work flow of correlative light electron microscopy (CLEM) imaging. (B) CLEM imaging of SOX2-positive cells at day-4 of CRS (n =13 cells per group). Representative images of autophagosomes (white arrows) in CRS group are shown at higher magnification. The number of autophagosomes was counted per 5m5m without counting the autophagosomes of whole cells using serial sections. Scale bars: 2 m. (C) Nes promoter-driven lentiviral expression of mRFP-EGFP-MAP1LC3B in vivo. Virus was injected 4days prior to CRS and analyzed at day 2 of CRS. Scale bar: 20 m. The graph on the right shows quantification of autophagosomes and autolysosomes (n =7 or 8 cells per group). (D and E) Cleaved CASP3 (c.CASP3) staining (D) and TUNEL assay (E) in the DG at day 7 of CRS. STS was injected 12h before analysis. Arrows indicate c.CASP3-positive cells. Scale bar: 100 m. ***P <0.001. n.s., not significant.

Figure 3.

Suppression of hippocampal neurogenesis, anxiety-like

Figure 3.

Suppression of hippocampal neurogenesis, anxiety-like and depressive behaviors, and spatial memory deficits induced

Suppression of hippocampal neurogenesis, anxiety-like and depressive behaviors, and spatial memory deficits induced by CRS are prevented in Atg7-NSC cKO mice. (A) Timeline for the measurement of the number of BrdU+ and RBFOX3+ double-positive NSCs. (B) Representative images of BrdU+ RBFOX3+ staining. Scale bar: 20 m. Arrow indicate RBFOX3+ BrdU+ double-positive cells. (C) Quantification of BrdU+ RBFOX3+-positive cells (n =6 or 7 per group). (D and E) Measurement of anxiety-like behaviors by the open field test (n =6 or 7 per group) (D) and elevated plus maze test (n =613 per group) (E). (F) Measurement of depressive behavior by the sucrose preference test (n =6). (G) Assessment of spatial working memory by the Y-maze test (n =68 per group). (H) Spatial learning and memory test using the acquisition phase (left graph) and probe test at day 6 (right graph) in MWM test (n =68 per group). TQ, target quadrant; AL, adjacent left; AR, adjacent right; OP, opposite. *P <0.05, **P <0.01, ***P <0.001.

Figure 4.

Atg7 deficiency prevents loss of

Figure 4.

Atg7 deficiency prevents loss of subtypes of NSCs in the adult DG and

Atg7 deficiency prevents loss of subtypes of NSCs in the adult DG and the activity of NSCs in neurosphere cultures. (A) Representative image of each subtype of NSCs in the SGZ of the DG. (band c) Quantification of GFAP+ SOX2+ (type 1), GFAP+ SOX2+ MKI67+ (active type 1), ASCL1+ SOX2+ (type 2a), DCX+ SOX2+ (type 2b), and DCX+ (type 3) NSCs 1 day (n =4 or 5 per group) (B) and 28days after CRS (n =5 or 6 per group) (C). (D) Representative image of neurospheres in culture for 7days. Scale bar: 40 m. (E) Quantification of neurospheres after 7days in cultures (n =35 per group). (F) Measurement of neurosphere size after 7days in cultures (n =35 per group). *P <0.05, **P <0.01, ***P <0.001.

Figure 5.

CORT-induced reduction of NSC number

Figure 5.

CORT-induced reduction of NSC number and hippocampal dysfuction is prevented in Atg7-NSC cKO

CORT-induced reduction of NSC number and hippocampal dysfuction is prevented in Atg7-NSC cKO mice. (A) Timeline of the CORT injection experiment. (B) Quantification of BrdU+ and SOX2+ cells (n =4 or 5 per group). **P <0.01 for the total SOX2+ cells ##P <0.01 for BrdU+ SOX2+ cells. (C) Nes promoter-driven lentiviral expression of mRFP-EGFP-MAPLC3B in SOX2+ cells in vivo. Scale bar: 20m. The graph on the right shows quantification of autophagosomes and autolysosomes (n =7 or 8 cells per group). (D) Timeline for the measurement of the number of BrdU+ RBFOX3+ doublepositive NSCs. (E) Quantification of BrdU+ RBFOX3+ double-positive cells (n =4 or 5 per group). (f) Elevated plus maze test (n =4 per group). (g) Y-maze test (n =4 per group). *P <0.05, **P <0.01, ***P <0.001.

Figure 6.

CORT treatment does not induce

Figure 6.

CORT treatment does not induce apoptosis or necroptosis in HCN cells. ( A

CORT treatment does not induce apoptosis or necroptosis in HCN cells. (A) Death rate of HCN cells after CORT treatment (n =4). Right, representative image of Hoechst and PI staining 48h after CORT treatment in HCN cells. (B) Nucleus condensation assay with Hoechst staining. (C) Immunostaining of cleaved CASP3 (c.CASP3). (D) Nuclear fragmentation assay by TUNEL staining. Scale bar: 40m for b-d. (E and F) Agarose gel electrophoresis of DNA fragmentation assay (E) and western blots of c.CASP3 (F) are representative of at least 3 experiments with similar results. All apoptotic markers were analyzed after CORT (200M for 48h, except western blotting analysis of c.CASP3 with 72h) or staurosporine (STS, 0.5M for 6h) treatment. (G) Effects of Z-VAD (25 M) or necrostatin-1 (NEC-1, 100 M) on HCN cell death after CORT treatment for 48h (n =3). (H) Western blotting analysis of the effects of Z-VAD (25 M) on autophagy flux after CORT treatment for 48h. The blots are representative of 3 experiments with similar results. *P <0.05, **P <0.01, ***P <0.001. n.s., not significant.

Figure 7.

CORT treatment induces ACD in

Figure 7.

CORT treatment induces ACD in HCN cells. ( A ) Representative EM images

CORT treatment induces ACD in HCN cells. (A) Representative EM images of HCN cells treated with CORT for 48h. Scale bar: 2 m. N, nucleus. The graph on the right shows quantification of autophagosomes (n =35 cells per group). (B) Western blotting analyses of MAP1LC3B and SQSTM1 levels after CORT treatment for 48h. The graphs on the right show quantification of MAP1LC3B-II (n =6) and SQSTM1 (n =4) after normalization to ACTB. (C) Analysis of autophagy flux using mRFP-EGFP-MAP1LC3B after CORT treatment for 48h. Scale bar: 10m. The graph on the right shows quantification of MAP1LC3B puncta (n =8). ***P <0.001 for the total MAP1LC3B puncta. #P <0.05, ##P <0.01 for yellow puncta. (D) Death rates of HCN cells after KO of Ulk1 (sgUlk1) or with stable knockdown of ATG7 (shAtg7), SQSTM1 (shSqstm1) or MAP1LC3B (shMap1lc3b) in comparison with control cells (sgCon or shCon) after CORT treatment for 48h (n =3). In all experiments, BafA1 (20nM) was added 1 h before cell harvest. *P <0.05, **P <0.01, ***P <0.001.

Figure 8.

SGK3 is critical for CORT-induced

Figure 8.

SGK3 is critical for CORT-induced cell death, but dispensable for apoptosis or necroptosis

SGK3 is critical for CORT-induced cell death, but dispensable for apoptosis or necroptosis in HCN cells. (A and B) Changes in the expression levels of SGK1, 2, and 3 following CORT treatment in HCN cells. mRNA levels after CORT treatment for 24h (n =3) (A). Western blotting analyses of protein levels (B). Blots are representative of 3 experiments with similar results. (C) KO of Sgk1 (sgSgk1) and Sgk2 (sgSgk2). (D) Death rates of sgSgk1 and sgSgk2 cells after CORT treatment for 48h (n =3). (E) KO of Sgk3 (sgSgk3). (F) Death rate of sgSgk3 cells (n =8). (G) Death rate of sgSgk3 cells after STS treatment (0.5 M) for 24h (n =3). (G) Death rate of sgSgk3 cells after H2O2 treatment (100M) for 6h (n =3). ***P <0.001. n.s., not significant.

Figure 9.

SGK3 mediates ACD in HCN

Figure 9.

SGK3 mediates ACD in HCN cells following CORT treatment. ( A ) Analysis

SGK3 mediates ACD in HCN cells following CORT treatment. (A) Analysis of autophagy flux by western blotting of MAP1LC3B. The graph shows quantification of MAP1LC3B-II after normalization to ACTB (n =5). (B) Analysis of autophagosome formation using mRFP-MAP1LC3B. Scale bar: 10m. The graph shows quantification of autophagosomes (n =4 or 5). (C) Time-course analysis of EGFP-ZFYVE1 puncta formation after CORT treatment using EGFP-ZFYVE1. Scale bar: 10m. The graph shows quantification of ZFYVE1 puncta (n =46). (D) Domain diagrams of SGK1, 2, and 3 showing the critical Arg90 residue in the Phox homology (PX) domain in SGK3. SGK1 has an incomplete PX domain [38]. (E) Effects of SGK3WT and SGK3R90A mutant on sgSgk3 cell death (n =4). EV, empty vector. (F) Analysis of autophagy flux by western blotting of MAP1LC3B in sgSgk3 cells transfected with SGK3WT or SGK3R90A mutant. The graph shows quantification of MAP1LC3B-II after normalization to ACTB (n =3). (G) Effects of the SGK3R90A mutation on the MAP1LC3B puncta formation and colocalization of SGK3 with MAP1LC3B. sgSgk3 cells were co-transfected with EGFP-tagged SGK3WT or SGK3R90A mutant with mRFP-MAP1LC3B. Scale bar: 10 m. In all experiments, BafA1 (20nM) was added 1 h before cell harvest. *P <0.05, ***P <0.001. n.s., not significant.

Figure 10.

SGK3 silencing attenuates NSC reduction

Figure 10.

SGK3 silencing attenuates NSC reduction in the DG after CRS. ( A )

SGK3 silencing attenuates NSC reduction in the DG after CRS. (A) Experimental design illustrating stereotaxic injection of AAV1/2-guide RNAs into DG of S-Cas9 KI mice. (B) Image of AAV1/2-sgSGK3-mCherry expression in the SGZ of DG. Scale bar, 100m. (C) KO of Sgk1 (AAV-sgSgk1) and Sgk3 (AAV-sgSgk3). (D) Timeline of the experiment. (E) Representative images of mCherry and SOX2 co-labeling in the SGZ of DG 3weeks after injection of AAV-sgSgk1 or AAV-sgSgk3 in S-Cas9 mice. Scale bar, 50m. (F) The graph shows quantification of SGK-mCherry and SOX2 double-positive cells. (n =5). ***P <0.001.

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This work was supported by the National Research Foundation of Korea (NRF) grants (2017R1A2B4004289, 2018M3C7A1056275), the KBRI basic research program (19-BR-01-08), and the DGIST Convergence Science Center Program (19-BD-04) of the Ministry of Science and ICT of Korea; National Research Foundation of Korea [2018M3C7A1056275]; National Research Foundation of Korea [2017R1A2B4004289]; Ministry of Science and ICT of Korea [19-BR-01-08]; Ministry of Science and ICT of Korea [19-BD-04].

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