Article Figures & Data
Figures
- Figure 1.
Astrocyte-specific deletion of Srf in SrfGFAPCKO mice. A, Representative images of immunostaining for SRF (red), S100β (green), and NeuN (blue) shows SRF expression in astrocytes (arrows) and neurons (arrowheads) in the cortex and striatum of control mice. SRF expression was seen only in the neurons but not in the astrocytes in SrfGFAPCKO mice mutant mice. A’, Representative higher magnification images of astrocytes and neurons from A showing SRF expression in control astrocytes and neurons and absent in mutant astrocytes. B, Quantification of SRF immunofluorescence in S100β+ astrocytes from A. At least 15–20 cells per mouse were analyzed (n = 3 mice). Astrocytes: cortex, control (16.49 ± 2.52), SrfGFAPCKO (4.80 ± 0.29); striatum, control (19.72 ± 3.179), SrfGFAPCKO (7.40 ± 1.47); neurons: cortex, control (24.33 ± 5.05), SrfGFAPCKO, control (28.47 ± 5.60); striatum, control (30.79 ± 1.40), SrfGFAPCKO (23.41 ± 3.09). C, Quantitative real-time PCR from whole-brain total RNA shows a significant decrease in Srf mRNA expression in the mutant mice relative to control mice; control (1.0 ± 0), SrfGFAPCKO (0.30 ± 0.14; n = 3 mice). D, Semi-quantitative PCR from whole-brain total RNA shows a significant decrease in Srf mRNA expression in the mutant mice. Rps29 expression served as the loading control (n = 3 mice). Control (1.04 ± 0.01), SrfGFAPCKO (0.24 ± 0.02; n = 3 mice). Scale bars: 50 μm (A) and 20 μm (A’); *p < 0.05, **p < 0.005, ***p < 0.0005; ns, not significant. Two tailed t test. Data are mean ± SEM.
- Figure 2.
Conditional deletion of SRF in astrocytes results in reactive gliosis. A, Representative images of immunostaining for GFAP and ALDH1L1 in three-week-old SrfGFAPCKO and control littermates shows higher expression of these markers in neocortex (Ctx) and hippocampus (Hc) of mutant mice as compared with control littermates. B, C, GFAP and S100β immunostaining also showed that the astrocytes in mutant mice were hypertrophic compared with control mice (n = 3 mice). D, Quantification of S100β+ astrocytes from (C). Cortex, control (76.58 ± 1.19), SrfGFAPCKO (50.25 ± 5.67); striatum, control (70.71 ± 3.61), SrfGFAPCKO (66.04 ± 3.72); hippocampus, control (11.90 ± 0.17), SrfGFAPCKO (10.88 ± 1.14; n = 3 mice). Scale bars: 100 μm (A) and 50 μm (B, C); ns, not significant. Two tailed t test. Data are mean ± SEM.
- Figure 3.
SRF ablation in astrocytes leads to widespread astrogliosis. A, Representative images of immunostaining for the astrogliosis marker, nestin in three-week-old SrfGFAPCKO and control littermates shows reactive astrocytes in cortex and hippocampus of Srf mutant mice but not in control littermates. B, Quantification of nestin fluorescence intensity shown in A. Cortex: control (0.0 ± 0.14), SrfGFAPCKO (771.9 ± 16.14), hippocampus: control (0.0 ± 8.15), SrfGFAPCKO (556.1 ± 38.56; n = 3 mice). C, Representative images showing co-immunostaining with GFAP and the gliosis marker, vimentin. There is little or no GFAP and vimentin expression in three-week control mice. In contrast, the astrocytes in mutant mice exhibit strong expression and colocalization of GFAP and vimentin. D, Quantification of fluorescence intensity in C. For GFAP, cortex, control (102.1 ± 5.28), SrfGFAPCKO (580.1 ± 62.10); hippocampus, control (228.0 ± 34.20), SrfGFAPCKO (638.1 ± 73.97); striatum, control (85.78 ± 1.83), SrfGFAPCKO (256.9 ± 7.73). For vimentin, cortex, control (2.93 ± 0.99), SrfGFAPCKO (674.10 ± 27.70); hippocampus, control (12.02 ± 12.02), SrfGFAPCKO (964.10 ± 11.79); striatum, control (2.33 ± 0.33), SrfGFAPCKO (633.50 ± 41.37). Shown here are neocortex, hippocampus (Hpc), and striatum (Str; n = 3 mice). Scale bars: 500 μm (A) and 50 μm (C); **p < 0.005, ***p < 0.0005, ****p < 0.0001. Two tailed t test. Data are mean ± SEM.
- Figure 4.
Absence of cell death in Srf mutant mice. A, Representative images of TUNEL staining of two-week-old SrfGFAPCKO mice and control littermates. Amplified view of boxed region is shown on right. Arrows show TUNEL+ cells while the arrowhead shows non-specific staining. B, Representative images of immunostaining for cleaved caspase-3 in the neocortex and hippocampus of two-week-old SrfGFAPCKO mice and control littermates. Amplified view of boxed region is shown on right. Arrows show cleaved caspase 3+ cells while the arrowhead shows non-specific staining. C, Quantification of TUNEL+ and cleaved caspase-3+ cells in neocortex and hippocampus (Hpc) shows the no significant difference in the number of dead cells between SrfGFAPCKO mice and control littermates. Caspase-3: cortex, control (2.20 ± 1.00), SrfGFAPCKO (1.45 ± 0.77); hippocampus, control (1.16 ± 0.65), SrfGFAPCKO (0.91 ± 0.79). TUNEL: cortex, control (0.37 ± 0.21), SrfGFAPCKO (0.08 ± 0.052); hippocampus, control (0.81 ± 0.21), SrfGFAPCKO (1.18 ± 0.25; n = 3 mice). D, Representative images of immunostaining for the proliferation marker, phH3 in three-week-old SrfGFAPCKO and their respective control mice showed no proliferating cells in the mutant mice. Immunostaining of P0.5 mouse brain section showed many phH3-positive cells and served as a control; n.s., not significant. Two-tailed t test. Data are mean ± SEM. Scale bars: 20 μm (A’, B’) and 200 μm (A, B) and 100 μm (D); n = 3 mice.
- Figure 5.
BBB is not compromised in SrfGFAPCKO mice. A, Schematic diagram showing experimental timeline of dextran fluorescein (DF) and HRP injection and tissue processing. B, 10-kDa TMR-dextran fluorescein (DF) tracer injection reveals normal architecture of cerebral vasculature in three- to five-week-old SrfGFAPCKO and their control littermates. C, Quantification of ratio of fluorescence intensity outside versus inside the blood vessel reveals no significant difference between SrfGFAPCKO mice and their control littermates, indicative of intact BBB. A stab-wounded brain served as a control to show BBB leakage. Control (0.21 ± 0.0), SrfGFAPCKO (0.18 ± 0.05), stab-wound (1.97 ± 0.26; n = 3 mice). D, Transcardial injection of 44-kDa HRP Type II in SrfGFAPCKO mice and their respective controls shows that HRP was restricted to the blood vessel lumen. E, Quantification of ratio of color intensity outside versus inside the blood vessel shows no significant difference between control and mutant mice (n = 3 mice). Control (0.16 ± 0.05), SrfGFAPCKO (0.20 ± 0.02), stab-wound (1.45 ± 0.08). Scale bars: 400 μm (B) and 200 μm (D); ns, not significant; ****p < 0.0001, one-way ANOVA, data are mean ± SD.
- Figure 6.
Persistent astrogliosis in SrfGFAPCKO mice. A, Immunostaining for GFAP in three-month-old, (3-mon) SrfGFAPCKO mice and control littermates shows widespread astrogliosis in all the brain regions. B, Quantification of color intensity in A. Cortex, control (107.2 ± 0.61), SrfGFAPCKO (176.4 ± 7.35); hippocampus, (Hpc), control (270.2 ± 9.11), SrfGFAPCKO (385.4 ± 16.14); striatum, control (151.4 ± 1.280), SrfGFAPCKO (260.6 ± 18.58; n = 3 mice). C, Representative images of immunostaining for GFAP in 12-month-old, (12-mon) SrfGFAPCKO mice and control littermates shows persistent astrogliosis in all the brain regions analyzed. Shown here are cortex, hippocampus (Hpc), and striatum. D, Quantification of relative color intensity in C. Quantification of color intensity in C; cortex, control (188.2 ± 2.85), SrfGFAPCKO (536.0 ± 26.03); hippocampus, control (356.3 ± 6.89), SrfGFAPCKO (591.0 ± 29.43); striatum: control (191.1 ± 4.83), SrfGFAPCKO (495.7 ± 80.51; n = 3 mice). E, Representative images of immunostaining for reactive astrogliosis marker, vimentin, in three- and 12-month-old SrfGFAPCKO mice and control littermates shows gliosis astrocytes only in the brains of Srf mutant mice. F, G, Relative fluorescent intensity of vimentin immunostaining in three-month-old (F) and 12-month-old (G) mice compared with control littermates. F, Cortex, control (11.52 ± 11.52), SrfGFAPCKO (317.1 ± 39.27); hippocampus: control (4.47 ± 3.11), SrfGFAPCKO (378.7 ± 8.17), striatum: control (5.43 ± 5.43), SrfGFAPCKO (110.8 ± 4.94). G, Cortex, control (8.22 ± 4.11), SrfGFAPCKO (475.7 ± 67.75); hippocampus, control (10.26 ± 10.26), SrfGFAPCKO (531.6 ± 31.96); striatum: control (17.30 ± 11.98), SrfGFAPCKO (157.4 ± 13.96; n = 3–4 mice). H, Immunostaining for the proliferation marker, phH3, in 12-month-old SrfGFAPCKO and control littermates showed no proliferating cells even at this age. Scale bar: 200 μm; *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. Two tailed t test. Data are mean ± SEM.
- Figure 7.
Microglial activation is seen along with astrogliosis in SrfGFAPCKO mice. A, Immunostaining for the microglia marker, Iba1, showed increased Iba1 expression in the neocortex and hippocampus of mutant mice as compared with the control littermates, suggesting activated microglia in mutant brain. B, Relative fluorescence intensity of Iba1 in the neocortex of Srf mutant mice compared with control littermates in A shows a significant increase in Iba1 expression in the Srf mutants indicative of microgliosis. Control (1.0 ± 0.0), SrfGFAPCKO (1.97 ± 0.19; n = 3 mice). C, Quantification of Iba1+ cells in three-week-old control and mutant mice. Cortex, control (29.08 ± 0.43), SrfGFAPCKO (34.4 ± 2.68); hippocampus (Hpc), control (28.64 ± 0.82), SrfGFAPCKO (33.80 ± 1.40); striatum, control (29.46 ± 1.20), SrfGFAPCKO (32.0 ± 0.42; n = 3 mice). D, Representative images of sections immunostained for microglial marker Iba1 in aged mice (three- and 12-month-old) in SrfGFAPCKO and control littermates show increased expression of Iba1 in the Srf mutants. Shown here is neocortex. E, Relative fluorescence intensity of Iba1 in the neocortex of Srf mutant mice compared with control littermates shows persistent microgliosis in the Srf mutants throughout adulthood. Three months, control (1.0 ± 0.0), SrfGFAPCKO (1.8 ± 0.22); 12 months, control (1.0 ± 0.0), SrfGFAPCKO (1.71 ± 0.17; n = 3 mice). F, G, Quantification of Iba1+ cells at three months (F), cortex, control (30.00 ± 0.62), SrfGFAPCKO (54.50 ± 6.87); hippocampus (Hpc), control (24.00 ± 4.25), SrfGFAPCKO (58.75 ± 9.00); striatum, control (29.25 ± 1.25), SrfGFAPCKO (32.75 ± 3.00); and 12 months (G) cortex, control (28.25 ± 0.57), SrfGFAPCKO (38.75 ± 2.49); hippocampus, (Hpc), control (27.58 ± 1.55), SrfGFAPCKO (36.75 ± 6.00); striatum, control (28.17 ± 0.88), SrfGFAPCKO (28.58 ± 1.17; n = 3 mice). Scale bars: 200 μm; **p < 0.005; ns, not significant. Two tailed t test. Data are mean ± SEM.
- Figure 8.
Prolonged gliosis in SrfGFAPCKO mice does not affect neuronal survival. A, Quantitative real-time PCR for A1, A2, pan-reactive astrocyte markers and neuroinflammatory genes shows expression of a greater number of A2 reactive genes in the brains of Srf mutant mice compared with A1 genes. The mutant astrocytes also exhibited a higher expression of neuroinflammatory genes, Il1β and Ccl2/Mcp-1 but not TNFα. Control (1.0 ± 0.0), SrfGFAPCKO, Cd109 (3.49 ± 0.62), Clcf1 (2.51 ± 0.52), Cd14 (2.82 ± 0.51), Ptgs2 (0.90 ± 0.25), Psmb8 (7.43 ± 0.83), H2T23 (2.20 ± 0.78), H2D1 (2.4 ± 0.65), Srgn (1.78 ± 0.39), Serpina3n (10.88 ± 1.86), Gfap (9.62 ± 0.84), Il1β (1.806 ± 0.16), Ccl2/Mcp-1 (13.52 ± 1.967), TNFα (3.35 ± 0.97; n = 3 mice). B, Representative images of immunostaining for the neuronal marker, NeuN shows normal structural integrity in three- to five-week-old SrfGFAPCKO mice compared with control littermates. C, D, Quantification of NeuN+ cells in three- to five-week-old (C) and 12-month-old (D) control and mutant mice shows no significant change in neuronal numbers in the mutant mice. C, Cortex, control (209.5 ± 12.10), SrfGFAPCKO (195.2 ± 11.18); CA1, control (78.25 ± 16.48), SrfGFAPCKO (148.4 ± 34.10); CA3, control (124.4 ± 24.47), SrfGFAPCKO (133.5 ± 37.20); striatum, control (220.8 ± 5.90), SrfGFAPCKO (205.8 ± 19.59; n = 3 mice). D, Cortex, control (208.4 ± 9.87), SrfGFAPCKO (190.0 ± 9.71); CA1, control (119.0 ± 22.36), SrfGFAPCKO (148.2 ± 12.23); CA3, control (101.0 ± 22.05), SrfGFAPCKO (127.8 ± 12.19); striatum, control (211.1 ± 5.14), SrfGFAPCKO (182.1 ± 51.89; n = 3 mice), Scale bar, 1 mm; *p < 0.05, **p < 0.005, ***p < 0.0005; ns, not significant. Two tailed t test. Data are mean ± SEM.
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