Sterol dysregulation in Smith–Lemli–Opitz syndrome causes astrocyte immune reactivity through microglia crosstalk

ABSTRACT Owing to the need for de novo cholesterol synthesis and cholesterol-enriched structures within the nervous system, cholesterol homeostasis is critical to neurodevelopment. Diseases caused by genetic disruption of cholesterol biosynthesis, such as Smith–Lemli–Opitz syndrome, which is caused by mutations in 7-dehydrocholesterol reductase (DHCR7), frequently result in broad neurological deficits. Although astrocytes regulate multiple neural processes ranging from cell migration to network-level communication, immunological activation of astrocytes is a hallmark pathology in many diseases. However, the impact of DHCR7 on astrocyte function and immune activation remains unknown. We demonstrate that astrocytes from Dhcr7 mutant mice display hallmark signs of reactivity, including increased expression of glial fibrillary acidic protein (GFAP) and cellular hypertrophy. Transcript analyses demonstrate extensive Dhcr7 astrocyte immune activation, hyper-responsiveness to glutamate stimulation and altered calcium flux. We further determine that the impacts of Dhcr7 are not astrocyte intrinsic but result from non-cell-autonomous effects of microglia. Our data suggest that astrocyte–microglia crosstalk likely contributes to the neurological phenotypes observed in disorders of cholesterol biosynthesis. Additionally, these data further elucidate a role for cholesterol metabolism within the astrocyte–microglia immune axis, with possible implications in other neurological diseases.

. Chromatographic and mass spectral parameters of sterols detected by GC/MS.Following isolation from astrocyte cultures, sterols were derivatized with trimethylsilyl (TMS) to ethers.After separation on a Rxi-5 Sil column (Restek, 43602), the above retention times and major fragment ions (m/z) were detected.

Fig. S2 .
Fig. S2.Dhcr7 astrocytes display reduced baseline calcium activity and response to ATP stimulation.(A) Trpc4 transcript expression is reduced in Dhcr7 astrocytes upon LPDS culture.Data show the mean  s.e.m. (*P0.05;two-tailed unpaired t-test.N = 3 biological replicates; each data point represents the average of three technical replicates).(B) Dhcr7 astrocytes exhibit reduced calcium response to ATP stimulation compared to control astrocytes.(C) Mean calcium intensity following ATP stimulation was reduced in Dhcr7 astrocytes.Data show the mean  s.e.m. (*P0.05;****P0.0001;two tailed unpaired t-test.N = 360 from three recordings per group; each data point represents the average intensity of the replicates over time).

Fig. S3 .
Fig. S3.Pharmacological inhibition of cholesterol synthesis and sterol profile changes.(A) Quantified GC/MS analyses of sterol content following dose response assay in control astrocytes treated with inhibitors of cholesterol biosynthesis.(B) Quantified GC/MS analyses of sterol content in control astrocytes treated with inhibitors of cholesterol biosynthesis demonstrates cholesterol depletion and accumulation of alternate sterols.Data show the mean  s.e.m.N = 3 biological replicates per group.

Fig. S4 .
Fig. S4.Pharmacological inhibition of cholesterol synthesis in human astrocytes induces morphological change consistent with reactivity.(A) Quantified GC/MS analyses of sterol content in human astrocytes treated with inhibitors of cholesterol biosynthesis shows substantial reduction of cholesterol levels accompanied by accumulation of sterol precursors (N = 3 biological replicates).(B) Hoechst nuclear counterstain and CellMask (blue) with GFAP (white) immunolabeling of human primary astrocytes.(C,D) Treatment of control human astrocytes with cholesterol biosynthesis inhibitors induces cellular hypertrophy and increased GFAP intensity.Scale bar: 100 m.Data show the mean  s.e.m. (****P 0.0001; one-way ANOVA and Dunnett's test versus LPDS control.N = 12 wells from three biological replicates, mean of 40 images per well).

Fig. S5 .
Fig. S5.Dhcr7 microglia exhibit enhanced lipid droplet accumulation.(A) Hoechst nuclear counterstain (blue) and BODIPY (green) immunolabeling of control and Dhcr7 primary microglia.(B,C) Analyses of control and Dhcr7 microglia shows increased BODIPY spot area and intensity in Dhcr7 microglia.Scale bar: 100 m.Data show the mean  s.e.m. (****P 0.0001; one-way ANOVA and Dunnett's test versus LPDS control.Average of 48 cells per group from three biological replicates, five images per replicate; each data point represents average spot area or intensity per cell).

Fig. S6 .
Fig. S6.Dhcr7 microglia induce lipid droplet accumulation in human primary astrocytes.(A) Hoechst nuclear counterstain and CellMask (blue) with BODIPY (green) immunolabeling of control and Dhcr7 microglia.(B,C) Control human astrocytes cocultured with control or Dhcr7 mouse microglia exhibit increased BODIPY spot area and intensity when cultured with Dhcr7 microglia.Scale bar: 100 m.Data show the mean  s.e.m. (*P 0.05; ****P 0.0001; one-way ANOVA and Dunnett's test versus astrocyte only control; Average of 64 cells per group from three biological replicates, five images per replicate; each data point represents average spot area or intensity per cell).