Secreted frizzled-related protein 3 regulates activity-dependent adult hippocampal neurogenesis

Abstract

Adult neurogenesis, the process of generating mature neurons from adult neural stem cells, proceeds concurrently with ongoing neuronal circuit activity and is modulated by various physiological and pathological stimuli. The niche mechanism underlying the activity-dependent regulation of the sequential steps of adult neurogenesis remains largely unknown. Here, we report that neuronal activity decreases the expression of secreted frizzled-related protein 3 (sFRP3), a naturally secreted Wnt inhibitor highly expressed by adult dentate gyrus granule neurons. Sfrp3 deletion activates quiescent radial neural stem cells and promotes newborn neuron maturation, dendritic growth, and dendritic spine formation in the adult mouse hippocampus. Furthermore, sfrp3 reduction is essential for activity-induced adult neural progenitor proliferation and the acceleration of new neuron development. Our study identifies sFRP3 as an inhibitory niche factor from local mature dentate granule neurons that regulates multiple phases of adult hippocampal neurogenesis and suggests an interesting activity-dependent mechanism governing adult neurogenesis via the acute release of tonic inhibition.

Figure 1. sfrp3 Expression and Regulation of Neurogenesis in the Adult Hippocampus

(A) RNA-seq quantification of sfrp1-5 expression in micro-dissected dentate gyri of adult wide-type (WT) mice 0 or 4 hours after a single ECS. Values represent mean ± SEM (n = 3; *: P < 0.01; one-way ANOVA). RPKM: reads per kilobase of exon per million fragments mapped. (B-C) sfrp3 expression in the adult mouse hippocampus. Shown in (B) are sample in situ images of sfrp3 expression in the whole hippocampus (top; scale bar: 100 µm) and dentate gyrus (bottom; scale bar: 20 µm). Note sfrp3 expression of in the granule cell layer (GCL), but not in the SGZ (bottom). ML: molecular layer. Shown in (C) are sample dual sfrp3 in situ and GFP immunostaining of the dentate gyrus of adult nestin-GFP mice. Scale bar: 20 µm. (D-F) Increased progenitor proliferation and newborn neuron number in the dentate gyrus of adult sfrp3 KO mice. In (D), animals were injected with BrdU and analyzed 2 hours later. Shown are sample projected confocal images of BrdU immunostaining (arrows) in the dentate gyrus of adult sfrp3 KO and WT littermate (top; scale bar: 50 µm) and stereological quantification of BrdU⁺ cells in the SGZ of adult WT, HET and KO littermates (bottom). Number associated with bar graphs indicates the number of animals examined. Values represent mean ± SEM (*: P < 0.01; n.s.: P > 0.1; one-way ANOVA). Shown in (E) are sample projected confocal images of MCM2 immunostaining and DAPI staining in the dentate gyrus (top; scale bar: 100 µm) and stereological quantification of MCM2⁺ cells. Values represent mean ± SEM (*: P < 0.05; student’s t-test). In (F), adult mice were injected with BrdU once daily for one week and examined four weeks after the first BrdU injection. Shown are sample projected confocal images of BrdU and NeuN immunostaining (top). Orthogonal views are shown to confirm co-localization of BrdU and NeuN. Scale bar: 50 µm. Also shown is stereological quantification of NeuN⁺BrdU⁺ mature newborn neurons (bottom). Values represent mean ± SEM (*: P < 0.01; student’s t-test). See also Figure S1 and Table S1.

Figure 2. Increased Activation of Quiescent Radial Glia-like Neural Stem Cells in the Dentate Gyrus of Adult sfrp3 Knockout Mice

(A-D) Sample confocal images of different types of GFP⁺ clones at 7 dpi. Adult nestin-CreER^T2+/−::Z/EG^f/+::sfrp3^−/− KO mice and nestin-CreER^T2+/−::Z/EG^f/+::sfrp3^+/+ control mice were injected with a single low dose of tamoxifen (62 mg/kg body weight, i.p.) and examined 7 days later. Shown are sample confocal images of immunostaining of GFP and GFAP for a quiescent clone with a single radial glia-like neural stem cell (RGL; A) and for activated clones with two RGLs (B), one RGL and one GFAP⁻ intemediate procursor cell (IPC; C), and one RGL and one GFAP⁺ astrocyte with bushy morphology (A; D). Scale bars: 10 µm. (E) Decreases RGL quiescence in the dentate gyrus of adult sfrp3 KO mice examined at 7 dpi. Values represent mean ± SEM (n = 6 WT and 7 KO mice; *: P < 0.01; student’s t-test) (F) No difference in percenatges of different types of activated clones in adult sfrp3 KO mice examined at 7 dpi. Values represent mean ± SEM. Same sets of animals as in (E) were used. See also Figure S2 and Table S1.

Figure 3. Regulation of Maturation, Dendritic Development and Spine Formation of Newborn Neurons in the Adult Hippocampus by sFRP3

(A-B) Accelerated new neuron maturation in the dentate gyrus of adult sfrp3 KO mice. Adult mice were injected with BrdU once daily for 1 week and analyzed 2 weeks after the first BrdU injection. Shown in (A) are sample confocal images of BrdU, DCX and NeuN immunostaining. Arrows point to BrdU⁺DCX⁺NeuN⁻ immature newborn neurons; arrowheads point to BrdU⁺DCX⁺NeuN⁺ more developed newborn neurons; and asterisks point to BrdU⁺DCX⁺NeuN⁻mature new neurons. Scale bar: 20 µm. Shown in (B) is a summary of the distribution of newborn neurons at different maturation stages. Values represent mean ± SEM (n = 9 animals each group; *: P < 0.01; one-way ANOVA). (C-G) Accelerated dendritic growth and spine formation of newborn neurons in the dentate gyrus of adult sfrp3 KO mice. Retroviruses expressing GFP were stereotaxically injected into the dentate gyrus of adult sfrp3 KO, HET and WT littermates. GFP⁺ neurons were examined at 14 dpi (C-D) or 21 dpi (E-H). Shown are sample projected confocal images of GFP⁺ newborn neurons at 14 dpi (C) and 21 dpi (E) and dendritic spines at 21 dpi (G). Scale bars: 20 mm. Also shown are cumulative distribution plots of total dendritic length, branch number and spine density of newborn neurons under different conditions (D, F and H). Each symbol represents data from a single GFP⁺ neuron (*: P < 0.01, Kolmogorov-Smirnov test; n = 3 animals each). See also Figure S3 and Table S1.

Figure 4. sfrp3 Reduction of sfrp3 Mediates Activity-dependent Adult Neurogenesis

(A) ECS decreases sfrp3 expression in the adult dentate gyrus. Shown are sample images of sfrp3 in situ in the adult hippocampus at different time after a single ECS (left; scale bar: 200 µm) and quantification by quantitative real-time PCR. Values are normalized to that of sham-treated WT animals at each time point and represent mean ± SEM (n > 3 animals for each time point; *: P < 0.01; student’s t-test). (B) Running activates dentate granule neurons and decreases sfrp3 expression. Shown are sample images of dual sfrp3 in situ and Arc immunostaining (left; scale bar: 20 µm) and quantification by quantitative real-time PCR (right). Values are normalized to that of sham-treated WT animals and represent mean ± SEM (n ≥ 3 animals for each time point; *: P < 0.01; student’s t-test). (C) sfrp3 deletion-induced increase of progenitor proliferation significantly occludes the ECS effect in the adult SGZ. Shown are schematic diagram of experimental design and stereological quantification of BrdU⁺ cells in the adult SGZ after a single ECS. Values represent mean ± SEM (*: P < 0.05; one-way ANOVA). (D) Exogenous sFRP3 blocks ECS-induced neural progenitor proliferation in adult WT mice. Shown are schematic diagram of experimental design and stereological quantification of BrdU⁺ cells in the adult SGZ under different conditions. Values represent mean ± SEM (*: P < 0.05; n.s.: P > 0.1; one-way ANOVA). (E) Running-induced increase of cell proliferation is attenuated in the adult sfrp3 KO mice. Same as in (C), except that mice were subjected to voluntary running. (F) sfrp3 deletion-induced dendritic growth of newborn neurons completely occludes the ECS effect. Same as in (C), except that dendritic growth of retrovirally labelled new neurons were analyzed at 14 dpi. See also Figure S4 and Table S1.