Figure 1.
KS expression was induced in the spinal cords of SOD1G93A mice at end stage.
(A) Lumbar spinal cord lysates (24 weeks of age) were subjected to immunoblotting using anti-KS antibody (5D4) (n = 5). β-actin was used as the internal loading control. (B) Lumber spinal cord sections obtained from SOD1G93A mice and their age-matched non-Tg littermates were stained with anti-KS antibody. The lower panels are the highly magnified images of the regions marked with squares. Bars, 200 µm in upper panels; 50 µm in lower panels.
Figure 2.
KS expression began before body weight loss.
(A) The lysates of the SOD1G93A lumbar spinal cords at 6, 12, 18, and 24 weeks of age were subjected to immunoblotting using the anti-KS antibody 5D4. β-actin was used as the internal loading control. A faint expression of KS was detected at 12 weeks. (B) The SOD1G93A lumber spinal cord section at 12 weeks was stained by anti-KS antibody. KS immunoreactivity was observed in the ventral horn. Bar, 200 µm.
Figure 3.
KS was expressed in a subpopulation of microglia.
(A-I) The SOD1G93A mice lumbar spinal cords at 18 weeks were stained by anti-KS (B, E, H), anti-Iba1 (microglia marker; A–C), anti-GFAP (astrocyte marker; D–F), and anti-NG2 (oligodendrocyte precursor marker; G–I) antibodies. Merged images are shown in C, F and I. Bars, 50 µm. Arrows, KS+Iba1+ cells; arrowheads, KS-Iba1+ cells. (J) A representative profile of KS expression in CD11b+ cells. The red line indicates a negative control that does not contain the primary antibody, and the blue line indicates a stained sample. (K) The quantitative data of mean fluorescence intensity (MFI) of KS (n = 3). Error bars, SE. **p<0.01. N.S., not significant. (L) The biosynthesis of KS. (M) mRNA expression of the enzymes involved in KS biosynthesis was examined by quantitative RT-PCR using SOD1G93A (gray columns) and their age-matched non-Tg (white columns) mice at 24 weeks. GAPDH was used as the internal control. Error bars, SE. ** p<0.01, * p<0.05 (n = 4). N.S., not significant.
Figure 4.
KS expression was detected in human ALS.
Human ALS spinal cord sections were stained by anti-Iba1 (A) and anti-KS antibodies (B). Arrows, Iba1+KS+ cells. Bars, 50 µm. The morphology of microglia was confirmed as anti-Iba1 antibody and DAB staining (D). The information of human ALS samples is shown in Figure 4E.
Figure 5.
KS expression was attenuated in the SOD1G93AGlcNAc6ST-1−/− mouse spinal cord.
KS expression in the spinal cords of SOD1G93A mice, age-matched non-Tg and SOD1G93AGlcNAc6ST-1−/− mice at 24 weeks was examined by immunoblotting (A) and immunohistochemical (B) analysis. KS was not expressed in the spinal cords of SOD1G93AGlcNAc6ST-1−/− mice. Bars, 200 µm. The number of microglia was examined by immunohistochemical (C) and immunoblotting (D) against Iba1 at 24 weeks. Bars, 200 µm. β-actin was used as the internal control (n = 4). E, The Iba1 band intensities were quantified by ImageJ. Error bars, SE. **p<0.01. N.S., not significant.
Figure 6.
Ablation of KS in microglia accelerated the early phase pathogenesis in a mouse model.
(A) The lifespan of SOD1G93A was 164.7±18.2 days (n = 43), while that of SOD1G93AGlcNAc6ST-1−/− was 158.7±13.2 days (n = 76) (p = 0.007). (B) Body weight loss of SOD1G93A began at 124.7±12.2 days, while it began at 118.4±11.9 days in SOD1G93AGlcNAc6ST-1−/− (p = 0.005). A decrease in rotarod performance at (C) 15 rpm and (D) 20 rpm of SOD1G93A began at 141.4±18.2 days and 121.6±24.5 days, respectively. In SOD1G93AGlcNAc6ST-1−/− mice, these onset times were accelerated to 125.2±14.2 (p<0.001) days and 112.5±18.1 days (p = 0.003), respectively.
Figure 7.
A subpopulation of KS-positive microglia was CD86-positive.
The spinal cord sections of SOD1G93A mice at 12, 18 and 24 weeks were stained with KS (A, E, I), M1 marker CD86 (B, F, J), and microglia marker Iba1 (C, G). The merged images are shown in (D, H, K). Arrows, KS+CD86+ microglia; arrowheads, KS+CD86− microglia. Bars, 50 µm. Flow cytometric analysis for CD11b+ cells at 24 weeks. Summaries of CD86 expression (L) and expressions of CD86 and KS (M) are shown. Gray columns, SOD1G93A mice; black columns, SOD1G93AGlcNAc6ST-1−/− mice. Error bars, SE. **p<0.01, *p<0.05 (n = 3), N.S., not significance.
Figure 8.
Transient enhancement of the expression of M2 markers was diminished in SOD1G93AGlcNAc6ST-1−/− mice.
The temporal mRNA expression profiles of M1 [CD86 (A), IL-1β (B), TNF-α (C) and NOX2 (D)] and M2 [Arginase1 (E), CD206 (F), Ym1 (G) and IL-4 (H)] markers were examined by quantitative RT-PCR. Gray columns, SOD1G93A mice; black columns, SOD1G93AGlcNAc6ST-1−/− mice. Error bars, SE. **p<0.01, *p<0.05 (n = 3).
Figure 9.
M1 microglia expanded as the disease progressed.
The spinal cord sections of SOD1G93A mice and SOD1G93AGlcNAc6ST-1−/− mice at 15 and 24 weeks were stained with KS (A, E, I, M), M1 marker CD86 (B, F, J, N), and microglia marker Iba1 (C, G, K, O). Arrows in A-H, KS+CD86+ microglia; arrows in J-P, KS–CD86+ microglia; arrowheads, KS+CD86− microglia. Bars, 50 µm.
Figure 10.
Expansion of M2 microglia during the early disease phase was suppressed in SOD1G93AGlcNAc6ST-1−/− mice.
The spinal cord sections of SOD1G93A mice and SOD1G93AGlcNAc6ST-1−/− mice at 15 and 24 weeks were stained with KS (A, E, I, M), M2 marker CD206 (B, F, J, N), and microglia marker Iba1 (C, G, K, O). Arrows, KS–CD206+ microglia; arrowheads, KS+CD206− microglia. Bars, 50 µm.
Figure 11.
CD206 was expressed in a subpopulation of microglia distinct from KS+CD86- cells.
Sequential thin sections were subjected to immunohistochemical analyses (A). The spinal cord sections of SOD1G93A mice at 15 weeks were stained with KS (B), CD86 (C) and CD206 (D). Arrows, KS+CD86− microglia; arrowheads, KS–CD206+ microglia. Bars, 50 µm.
Table 1.
The primer sequences determined using genotyping of mice.
Table 2.
The primer sequences using quantitative RT-PCR of M1 and M2 markers.