Research ArticleNEUROSCIENCE

Dynamic Fas signaling network regulates neural stem cell proliferation and memory enhancement

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Science Advances  22 Apr 2020:
Vol. 6, no. 17, eaaz9691
DOI: 10.1126/sciadv.aaz9691
  • Fig. 1 Development and validation of the optogenetically activatable Fas receptor.

    (A) Schematic diagram of optoFAS and downstream signaling pathways. NFκB, nuclear factor κB. (B) Representative confocal images of apoptosis induced in optoFAS-transfected HeLa cells exposed to light stimulation, as compared to optoFAS-transfected cells incubated in the dark and Lyn-cyFAS-EGFP–transfected cells subjected to light stimulation. Scale bar, 50 μm. (C) Quantification of the apoptotic induction of HeLa cells over time. OptoFAS light, cells expressing optoFAS subjected to continuous blue light of 5 μW/mm2; OptoFAS dark, the same cells incubated in dark; FKBP/FRB + Rap, cells transfected with Lyn-cyFAS-FKBP-EGFP and Lyn-cyFAS-FRB-mCherry treated with 500 nM rapamycin; FKBP/FRB − Rap, the same cells without rapamycin treatment; sFasL, treatment with soluble Fas ligand (100 ng/ml); cisplatin, treatment with cisplatin (10 μg/ml; n ≥ 80 cells per group). (D) Representative confocal images of optoFAS- and caspase-3 biosensor–transfected HeLa cells undergoing apoptosis, showing the activation of caspase-3. Scale bar, 20 μm. (E) Representative confocal images of optoFAS- and caspase-8 biosensor–transfected HeLa cells undergoing apoptosis, showing the activation of caspase-8. Scale bar, 20 μm. (F) Quantification of caspase-3 biosensor activity for the cells shown in (D). (G) Quantification of caspase-8 biosensor activity for the cells shown in (E). a.u., arbitrary units. (H) Activation of JNK in optoFAS-transfected cultured hippocampal neurons at DIV (days in vitro) 7 with and without illumination, as revealed by the JNK-KTR sensor (n = 20 cells were included in the both light and dark groups). (I) Representative immunocytochemical (ICC) staining images of optoFAS-transfected cells with or without light stimulation, showing pS6 expression. Scale bar, 50 μm. (J) Quantification of the data shown in (I). Data are given as means ± SEM; n ≥ 100 cells per each group. Two-way analysis of variance (ANOVA) was used for statistical analysis. ****P < 0.0001. ns, not significant.

  • Fig. 2 Optogenetic activation of Fas reveals a dynamic signaling network in the DG.

    (A) A schematic representation and timeline showing the viral injection and experimental procedures. (B) A representative image showing the expression of AAV-Nestin-Cre and AAV-hSyn-DIO-optoFAS in the DG. Scale bar, 200 μm. DAPI, 4′,6-diamidino-2-phenylindole. (C) Representative images of changes in the levels of pS6 and pErk in the optoFAS-transduced DG as the duration of illumination increased. Inset shows a representative focus area. Scale bars, 100 μm and 50 μm (inset). (D) Quantification of the pS6+ cells in (C). Data are presented as means ± SEM; n ≥ 4 mice were included under each condition. One-way ANOVA was used for statistical analysis. ****P < 0.0001 and *P < 0.05. (E) Quantification of the pErk+ cells in (C). Data are presented as means ± SEM; n ≥ 4 mice were included under each condition. One-way ANOVA was used for statistical analysis. ****P < 0.0001 and *P < 0.05. (F) Representative images showing the colocalization of GFP+ cells and pS6+ cells. Scale bar, 20 μm. (G) Quantification of (F). Data are presented as means ± SEM; n = 6 mice. A single section per mouse was randomly selected. n ≥ 20 pS6+ cells were included in each section. (H) Representative images showing the lack of colocalization of GFP+ cells and pErk+ cells. Scale bar, 20 μm. (I) Quantification of data shown in (H). Data are presented as means ± SEM; n = 6 mice. A single section per mouse was randomly selected. n ≥ 20 pErk+ cells were included in each section. (J) pErk+ cells in the SGZ counterstained with the neural stem cell markers, SOX2 (top) and DCX (bottom). Arrowheads indicate cells with colocalizing signals. Scale bars, 50 μm. (K) The proportion of either SOX2+ or DCX+ cells among all pErk+ cells. Data are presented as means ± SEM; n = 5 mice. A single section per mouse was randomly selected. n ≥ 20 pErk+ cells were included in each section. (L) A schematic diagram and timeline showing the rapamycin-induced blockade of the mTOR pathway in vivo. i.p., intraperitoneal. (M) Representative images of the effect of mTOR blockage on pS6 and the pErk level. Scale bar, 100 μm. (N) Quantification of the pS6+ cells in (M, top row). Data are presented as means ± SEM; n = 5 mice per group, four sections per mouse were randomly selected. An unpaired two-tailed t test was used for statistical analysis. ****P < 0.0001. (O) Quantification of the pErk+ cells in (M, bottom row). Data are presented as means ± SEM; n = 5 mice per group, four sections per mouse were randomly selected. An unpaired two-tailed t test was used for statistical analysis. ****P < 0.0001.

  • Fig. 3 Optogenetic stimulation of Fas in immature neurons induces BDNF secretion, working as a paracrine mediator.

    (A) A schematic diagram and timeline for the viral transduction and light stimulation of cultured hippocampal neurons. (B to D) Quantitative real-time polymerase chain reaction (qRT-PCR) results for BDNF (B), IGF-1 (C), and IL-6 (D) in optoFAS-transduced neurons exposed to light stimulation, GFP-transduced neurons exposed to light stimulation, and GFP-transduced neurons treated with soluble Fas ligand. Data are presented as means ± SEM; n = 3 per group. One-way ANOVA was used for statistical analysis. **P < 0.01, ***P < 0.001, and ****P < 0.0001. (E) A schematic illustration and timeline for the viral transduction, rapamycin treatment, and light stimulation of cultured hippocampal neurons. (F and G) qRT-PCR results for BDNF (F) and IGF-1 (G) in optoFAS-transduced neurons treated with rapamycin and exposed to light stimulation and GFP-transduced neurons treated with soluble Fas ligand. Data are presented as means ± SEM; n = 3 per group. Two-way ANOVA was used for statistical analysis.**P < 0.01. (H) The results of BDNF ELISA for the supernatants of optoFAS-transduced neurons exposed to light stimulation, GFP-transduced neurons exposed to light stimulation, and GFP-transduced neurons treated with Fas ligand. Data are presented as means ± SEM; n ≥ 3 per group. Two-way ANOVA was used for statistical analysis. ****P < 0.0001. (I) A schematic illustration showing how immature neurons in the Bdnfflox/flox mouse are targeted to acquire deficits in BDNF transcription via transduction of AAV-Nestin-Cre and AAV-hSyn-DIO-optoFAS. (J) Representative images of pS6 (top) and pErk (bottom) IHC staining of a Bdnfflox/flox mouse transduced by AAV-Nestin-Cre and AAV-hSyn-DIO-optoFAS and subjected to illumination. Inset shows a representative focus area. Scale bars, 100 μm and 50 μm (inset). (K) Quantification of the pS6+ cells in (J) and comparison to that of Bdnf+/+ mice. Data are presented as means ± SEM; n = 5 mice. Four sections per mouse were randomly selected. An unpaired two-tailed t test was used for statistical analysis. (L) Quantification of the pErk+ cells in (J) and comparison to that in Bdnf+/+ mice. Data are presented as means ± SEM; n = 5 mice. Four sections per mouse were randomly selected. An unpaired two-tailed t test was used for statistical analysis. ***P < 0.001. (M) A schematic diagram and timeline for viral injection and experimental procedure for qRT-PCR of mouse hippocampal tissue. (N) Total BDNF mRNA expression level in brains from optoFAS-transduced mice exposed to light stimulation and from exercised mice. Data are presented as means ± SEM; n = 3 per group. One-way ANOVA was used for statistical analysis. ****P < 0.000 and ***P < 0.001. (O) mRNA expression levels for the splicing variants of BDNF in the optoFAS-transduced mouse brain and exercised mouse brain. Data are expressed as means ± SEM; n = 3 per group. (P) Timeline for viral injection and experimental procedure for qRT-PCR of mouse hippocampal tissue following exposure to repetitive stimulation. (Q) Total BDNF mRNA expression level in optoFAS-transduced hippocampus subjected to single or repetitive light stimulation. Data are presented as means ± SEM; n = 3 per group. One-way ANOVA was used for statistical analysis. *P < 0.05 and **P < 0.01. (R) The mRNA expression levels of BDNF II, IV, VI, and VIII in optoFAS-transduced hippocampus subjected to single or repetitive light stimulation. Data are presented as means ± SEM; n = 3 per group. One-way ANOVA was used for statistical analysis. ****P < 0.0001.

  • Fig. 4 Repetitive activation of Fas induces adult hippocampal neurogenesis.

    (A) A schematic illustration and timeline of the experiment used to evaluate neural stem cell proliferation following a single or repetitive light stimulation of the optoFAS-transduced mouse brain. (B) Representative images of neural stem cell proliferation upon light stimulation, as assessed by BrdU IHC staining (left column; inset, a representative focus area) and images of optoFAS expression in each case (right column). Scale bars, 100 μm and 50 μm (inset). (C) BrdU+ cells in the SGZ following five rounds of light stimulation, in sections counterstained for SOX2 and DCX (inset shows the boxed region; arrowheads represent cells with colocalizing signals). Scale bars, 100 μm and 20 μm (inset). (D) Quantification of the BrdU+SOX2+ or BrdU+DCX+ cells in the DG following single or repetitive light stimulation of optoFAS-transduced mouse brain. Data are presented as means ± SEM; n = 5 mice under each condition. One-way ANOVA was used for statistical analysis. ***P < 0.001 and ****P < 0.0001. (E) Timeline of the experiment for evaluating adult neurogenesis upon repetitive optoFAS stimulation. (F) Representative images of BrdU+ cells counterstained for calretinin. Right: An enlargement of the boxed region in the left image; arrowheads indicate cells with colocalizing signals. Scale bars, 50 μm (left) and 20 μm (right). (G) Quantification of the data shown in (F). Data are presented as means ± SEM; n = 5 mice under each condition. An unpaired two-tailed t test was used for statistical analysis. ***P < 0.001. (H) Timeline of the experiment used to evaluate neural stem cell proliferation upon blockage of the mTOR pathway in optoFAS-transduced mouse brain. (I) Representative images of BrdU+ cells in optoFAS-transduced DG subjected to rapamycin treatment (inset shows the boxed region). Scale bars, 100 μm and 50 μm (inset). (J) Quantification of the data shown in (I). Data are presented as means ± SEM; n = 5 mice under each condition. One-way ANOVA was used for statistical analysis. ****P < 0.0001 and ***P < 0.001.

  • Fig. 5 Repetitive activation of the Fas signaling network induces transient memory increase.

    (A) A schematic illustration and timeline for the behavioral tests used to assess optoFAS-transduced mice with light stimulation. (B) Spontaneous alteration on Y-maze tests of the optoFAS-transduced mice exposed to single or repetitive light stimulation. Data are presented as means ± SEM; n = 5 to 6 mice per group. One-way ANOVA was used for statistical analysis. **P < 0.01. (C) Total entries on Y-maze tests among optoFAS-transduced mice exposed to a single or repetitive light stimulation. Data are presented as means ± SEM; n = 5 to 6 mice per group. One-way ANOVA was used for statistical analysis. (D) Timeline for the behavioral tests of optoFAS- or GFP-transduced mice given prolonged incubation after light stimulation. (E) Spontaneous alteration on Y-maze tests among optoFAS- or GFP-transduced mice subjected to five rounds of light stimulation followed by varying durations of incubation. Data are presented as means ± SEM; n = 5 mice per group. One-way ANOVA was used for statistical analysis. *P < 0.05. (F) Total entries on Y-maze tests among optoFAS- or GFP-transduced mice subjected to five rounds of light stimulation, followed by varying durations of incubation. Data are presented as means ± SEM; n = 5 mice per group. One-way ANOVA was used for statistical analysis. (G) Timeline for the behavioral tests of optoFAS-transduced mice exposed to light stimulation in the presence of mTOR pathway blockade. (H) Spontaneous alteration on Y-maze tests among optoFAS-transduced mice subjected to repetitive stimulation and rapamycin treatment. Data are presented as means ± SEM; n = 5 to 6 mice per group. One-way ANOVA was used for statistical analysis. **P < 0.01. (I) Total entries on Y-maze tests among optoFAS-transduced mice subjected to repetitive stimulation and rapamycin treatment. Data are presented as means ± SEM; n = 5 to 6 mice per group. One-way ANOVA was used for statistical analysis. (J) Timeline for the behavioral tests of optoFAS-transduced mice exposed to light stimulation in the presence of BDNF/TrkB blockade. (K) Spontaneous alteration on Y-maze tests among optoFAS-transduced mice subjected to repetitive stimulation and ANA-12 treatment. Data are presented as means ± SEM; n = 5 mice per group. One-way ANOVA was used for statistical analysis. *P < 0.05. DMSO, dimethyl sulfoxide. (L) Total entries on Y-maze tests among optoFAS-transduced mice subjected to repetitive stimulation and ANA-12 treatment. Data are presented as means ± SEM; n = 5 mice per group. One-way ANOVA was used for statistical analysis.

  • Fig. 6 Summary of molecular and cellular dynamics of Fas signaling in the adult hippocampal DG upon prolonged and repetitive activation.

    (A) Summary of the molecular dynamics of Fas signaling in the DG upon optogenetic Fas activation. Transient Fas activation in immature neurons of the DG induces activation of the mTOR pathway in these cells. Prolonged activation results increases pErk in neural stem cells through BDNF secreted from Fas-activated immature neurons. NSC, neural stem cell; IN, immature neuron; MN, mature neuron. (B) Summary of the histological and behavioral outcomes observed following repetitive activation of Fas signaling in the DG. Repetitive activation of the Fas–mTOR (immature neuron)–BDNF VI–pErk (neural stem cell) paracrine pathway in the DG induces the proliferation of neural stem cells and transiently increases spatial working memory.

Supplementary Materials

  • Supplementary Materials

    Dynamic Fas signaling network regulates neural stem cell proliferation and memory enhancement

    Seokhwi Kim, Nury Kim, Jinsu Lee, Sungsoo Kim, Jongryul Hong, Seungkyu Son, Won Do Heo

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