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, 17 (15), 2257-64

Sphingosine Kinase 1/S1P Receptor Signaling Axis Controls Glial Proliferation in Mice With Sandhoff Disease

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Sphingosine Kinase 1/S1P Receptor Signaling Axis Controls Glial Proliferation in Mice With Sandhoff Disease

Yun-Ping Wu et al. Hum Mol Genet.

Abstract

Sphingosine-1-phosphate (S1P) is a lipid-signaling molecule produced by sphingosine kinase in response to a wide number of stimuli. By acting through a family of widely expressed G protein-coupled receptors, S1P regulates diverse physiological processes. Here we examined the role of S1P signaling in neurodegeneration using a mouse model of Sandhoff disease, a prototypical neuronopathic lysosomal storage disorder. When sphingosine kinase 1 (Sphk1) was deleted in Sandhoff disease mice, a milder disease course occurred, with decreased proliferation of glial cells and less-pronounced astrogliosis. A similar result of milder disease course and reduced astroglial proliferation was obtained by deletion of the gene for the S1P(3) receptor, a G protein-coupled receptor enriched in astrocytes. Our studies demonstrate a functional role of S1P synthesis and receptor expression in astrocyte proliferation leading to astrogliosis during the terminal stages of neurodegeneration in Sandhoff disease mice. Because astrocyte responses are involved in many types of neurodegeneration, the Sphk1/S1P receptor signaling axis may be generally important during the pathogenesis of neurodegenerative diseases.

Figures

Figure 1.
Figure 1.
Alteration of S1P metabolic enzymes and S1P receptor expression in the spinal cord of Sandhoff disease mice. (A) The pathway of S1P metabolism and receptor signaling. mRNA expression of the genes involved in S1P synthesis and degradation (Sphk1, Sphk2, Sgpp1, Sgpp2, Sgpl1) (B) and S1P receptors (S1P1,2,3,4,5) (C) in the spinal cord of 8- and 16-week-old Hexb−/− and Hexb+/+ mice as determined by real-time PCR (n = 3–5 per group, *P < 0.05; **P < 0.01). Levels of mRNA are expressed relative to the mRNA levels in Hexb+/+ mice (set at one). Measurement of Sphk activity (D) and S1P levels (E) in spinal cord of 16-week-old Hexb+/+Sphk1+/+, Hexb+/+Sphk1−/−, Hexb−/−Sphk1+/+ and Hexb−/−Sphk1−/− mice. The Sphk assay was performed in the presence of Triton X-100 to maximize the detection of Sphk1 activity. TLC analysis of [32P]-S1P formed by Sphk activity present in spinal cord samples (D) and S1P extracted from homogenates of spinal cord (E) from 16-week-old mice. The bands for S1P and the origin (Ori) are shown. The quantitated data represent mean values±SEM (n = 3 per group, *P < 0.05).
Figure 2.
Figure 2.
Localization of Sphk1 expression in spinal cord. Spinal cord sections of Hexb+/+ (A) and Hexb−/− (B) mice at 16 weeks of age were stained with antibody against Sphk1 and processed for immunohistochemical detection. Arrows indicate the ventral horn motor neurons. Arrowheads indicate other Sphk1-positive cells. Scale bars, 50 µm. Double immunofluorescence staining localizing Sphk1 (green) and GFAP (red, to identify astrocytes) of Hexb+/+ (C) and Hexb−/− (D) mice at 16 weeks of age. Arrows in D indicate plasma membrane-localized Sphk1. Arrowheads indicate astrocytes that are also Sphk1-positive. Scale bar, 50 µm.
Figure 3.
Figure 3.
Sphk1 deletion lengthens lifespan and improves motor function in Hexb−/− mice. (A) Physical condition of double null Hexb−/−Sphk1−/− mouse at 15 weeks of age compared with littermate single null Hexb−/− mouse. Note more severe wasting of the hindquarters in single null Hexb−/− mouse. (B) The reduction of body weight of Hexb−/−Sphk1−/− mice was delayed compared with Hexb−/−Sphk1+/+ mice (n = 10 per group, *P < 0.05). (C) Kaplan–Meier curves of the survival of Hexb−/−Sphk1−/− mice (n = 8) compared with Hexb−/−Sphk1+/− (n = 11) and Hexb−/−Sphk1+/+ mice (n = 12), all on C57BL6 background (P = 0.0013; log-rank test). (D) Motor function of Hexb+/+Sphk1+/+, Hexb−/−Sphk1+/+, Hexb+/+Sphk1−/− and Hexb−/−Sphk1−/− mice, all on C57BL6 background, was tested by rotorod performance. Mean±SEM time to fall off the rotorod (n = 10 per group). The statistical analysis was performed between Hexb−/−Sphk1+/+ and Hexb−/−Sphk1−/− mice, *P < 0.01.
Figure 4.
Figure 4.
Sphingolipid metabolic enzyme expression and glycolipid storage in mutant mice. (A) Expression of Sphk2, Sgpp1, Sgpp2 and Sgpl1 mRNA by real-time PCR in spinal cord of 8- and 16-week-old mice (n = 3–5 per group, *P < 0.05). Levels of mRNA are expressed relative to the mRNA levels in Hexb+/+ mice (set at a value of one). TLC analysis of spinal cord acidic (B) and neutral (C) glycolipids in 16-week-old mice. The GM2 and GA2 bands are boxed.
Figure 5.
Figure 5.
Deletion of Sphk1 and S1P3 reduces cell proliferation in the spinal cord of Sandhoff mice. (A) The numbers of cell nuclei (arrows) in the ventral spinal cord in 16-week-old Hexb−/−Sphk1−/− and Hexb−/−S1P3−/− mice were reduced compared with Hexb−/− mice, as observed in H&E-stained sections (black line demarcates the ventral spinal cord) and in Ki67-immunohistochemically stained paraffin sections. Scale bar, 250 µm. (B) Quantitation of Ki67-positive cells from 10 randomly selected section for each mouse (n = 3 per group). The data represent mean values±SEM, with the comparisons being Hexb−/− versus Hexb−/−Sphk1−/− and Hexb−/− versus Hexb−/−S1P3−/−; **P < 0.01. (CF) Expression of vimentin, GFAP, cyclin D1 and S1P3 receptor mRNA by real-time PCR in spinal cord of 16-week-old mice (n = 3–5 per group). The data represent mean values±SEM, with the comparisons being Hexb−/− versus Hexb−/−Sphk1−/− and Hexb−/− versus Hexb−/−S1P3−/−; *P < 0.05. Insets in (C) and (D) show examples of Ki67-positive cells that were also positive for vimentin and GFAP, respectively. Scale bar, 20 µm.
Figure 6.
Figure 6.
Astrogliosis in mutant mice. (A) Comparison of reactive astrocytes in the ventral spinal cord by GFAP immunofluorescent staining in mice of different genotypes at 16 weeks of age. Note the decreased astrogliosis in Hexb−/−Sphk1−/− or Hexb−/−S1P3−/− mice compared with single null Hexb−/− mice. (B) High-power image showing that the processes of astrocytes were denser in single null Hexb−/− mice than in double null Hexb−/−S1P3−/− mice, even though the soma of astrocytes were hypertrophied (asterisks indicate the motor neurons). Scale bars, 100 µm.
Figure 7.
Figure 7.
Deletion of S1P3 lengthens lifespan and improves motor function of Sandhoff disease mice. (A) S1P3 receptor and GFAP co-immunofluorescent staining in spinal cord of 16-week-old Hexb−/− mice demonstrates that many of the receptor-positive cells are also positive for GFAP. Scale bar, 50 µm. (B) Kaplan–Meier curves of the survival for Hexb−/−S1P3−/− mice (n = 19) compared with Hexb−/−S1P3+/− (n = 15) and Hexb−/−S1P3+/+ mice (n = 12), all on C57Bl6 background (P = 0.0001; log-rank test). (C) Motor function of Hexb+/+S1P3+/+, Hexb−/−S1P3+/+, Hexb+/+S1P3−/− and Hexb−/−S1P3−/− mice was tested. Mean±SEM time to fall off the rotorosd (n = 10 per group), all on C57Bl6 background. The statistical analysis was performed between Hexb−/−S1P3+/+ and Hexb−/−S1P3−/− mice, *P < 0.01.

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