Journal: Acta Pharmaceutica Sinica. B

Article Title: Neuron stem cell NLRP6 sustains hippocampal neurogenesis to resist stress-induced depression

doi: 10.1016/j.apsb.2023.03.010

Figure Lengend Snippet: NLRP6 abundantly expressed in NSCs supports hippocampal NSC proliferative capacity. (A) Primary cells including NSCs, neurons, astrocytes and microglia were isolated from hippocampus of mice. Western blotting was used to detect the protein level. Right: densitometry analysis of NLRP6 protein in each cell ( n = 2). (B) RNAscope and immunofluorescence co-labeled the coronal section of the hippocampus. In hippocampus of WT mice at 8-week-old, single confocal planes labeled with Nlrp6 mRNA probes (cyan) showed co-expression of NESTIN (green) and GFAP (red) protein simultaneously, but not co-expression with GFAP positive astrocytes individually (Scale bar, 150 and 20 μm, respectively; rectangular area 1, rich in NSCs; rectangular area 2, rich in astrocytes; yellow arrowheads, NSCs; red arrowheads, astrocytes). (C) Representative maximum-intensity projection of a confocal image z-stack was shown in mice, labeled with NESTIN (green) and GFAP (red) in hippocampus (Scale bar, 20 μm). (D) NESTIN and GFAP co-labeled NSCs were quantified ( n ≥ 5). (E) Coronal section staining of mice at 16-week-old in hippocampus, representative maximum-intensity projection of a confocal image z-stack of mice was shown, labeled with hoechst (blue), NESTIN (green), GFAP (red), and KI67 (magenta) (the image below is an enlargement of the rectangular area in the picture above, scale bar, 150 or 20 μm). (F) NESTIN, GFAP, and KI67 positive NSCs were quantified ( n ≥ 5). (G) The relative mRNA level was detected by qRT-PCR in hippocampus of mice at 16-week-old ( n ≥ 8). (H) Primary NSCs were isolated and cultured, and the diameter of neurospheres was detected under the light microscope ( n ≥ 6). (I) Representative confocal images of NSCs neurospheres labeled with hoechst (blue), NESTIN (red), and KI67 (green) are shown (scale bar, 50 μm). (J) KI67 positive cells accounted for the percentage of total cells in neurospheres ( n ≥ 4). (K) Cell proliferation was detected by CCK-8 assay ( n = 10). NSCs, neural stem cells; GFAP, glial fibrillary acidic protein; IBA1, ionized calcium-binding adapter molecule 1; Cdk, Cyclin-dependent kinase; CCK-8, cell counting kit-8. Data are represented as mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: 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).

Techniques: Isolation, Western Blot, RNAscope, Immunofluorescence, Labeling, Expressing, Staining, Quantitative RT-PCR, Cell Culture, Light Microscopy, CCK-8 Assay, Binding Assay, Cell Counting