7-Dehydrocholesterol-derived oxysterols cause neurogenic defects in Smith-Lemli-Opitz syndrome
- Department of Medicinal Chemistry, University of Washington, United States
Figures
Figure 1 with 1 supplement
Loss of Dhcr7 alleles causes decreased proliferation and increased neurogenesis in murine and human neural progenitor cells.
(A) RT-PCR for Dhcr7 mRNA in the E11.5 to P0 cortex. β-actin mRNA was used as the loading control. (B) qRT-PCR for Dhcr7 mRNA in the E11.5 to E18.5 cortex. Data is expressed as fold change over …
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Figure 1—source data 1
Related to Figure 1A.
RT-PCR for Dhcr7 mRNA in the E11.5 to P0 cortex. β-actin mRNA was used as loading control.
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig1-data1-v2.pdf
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Figure 1—source data 2
Related to Figure 1C.
Western blot of Dhcr7 in total cortical lysates from E13.5 to P0. The blot was re-probed for Erk1/2 as a loading control.
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig1-data2-v2.pdf
Figure 1—figure supplement 1
Characterization of the pluripotency of SLOS-derived human iPSCs.
Related to Figure 1. Pluripotency of SLOS patient derived human iPSC lines, 3044 and 5788 were characterized. (A) Representative images of established SLOS patient derived human iPSC lines (3044 and …
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Figure 1—figure supplement 1—source data 1
Related to Figure 1—figure supplement 1B.
RT-PCR analysis of hES cell marker genes in human iPSCs derived from SLOS patients (3044 and 5788) and healthy individual (emhf2).
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig1-figsupp1-data1-v2.pdf
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Figure 1—figure supplement 1—source data 2
Related to Figure 1—figure supplement 1D.
RT-PCR analyses and Western blot analyses of differentiation markers for the three germ layers.
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig1-figsupp1-data2-v2.pdf
Figure 2 with 2 supplements
Cholesterol precursor 7-DHC and 7-DHC-derived Oxysterols are accumulated in Dhcr7-/- mouse embryonic cortex.
(A) Chemical structures of 7-DHC-derived oxysterols. LC-MS/MS analysis of (B) cholesterol and its precursors and (C) 7-DHC and cholesterol-derived oxysterols in Dhcr7+/+ and Dhcr7-/- embryonic …
Figure 2—figure supplement 1
Cholesterol biosynthesis pathway, the enzymes involved in the synthesis and the diseases associated with the defective enzymes.
Related to Figure 2.
Figure 2—figure supplement 2
Cholesterol precursor 7-DHC and 7-DHC-derived Oxysterols are accumulated in SLOS-derived human iPSCs and NPCs.
Related to Figure 2. SLOS patient derived hiPSCs and NPCs (3044 and 5788) along hiPSCs and NPC from healthy individual (emhf2) were cultured and used for LC-MS/MS analysis sterols and oxysterols. …
Figure 3 with 1 supplement
Knockdown of Dhcr7 causes increased neurogenesis and depletion of cycling precursors in murine and human NPC cultures.
(A) Western blot for Dhcr7 in 293T cells transfected with control or individual murine Dhcr7 shRNAs. The blot was re-probed for Erk1/2 as a loading control. (B and C) Mouse cortical precursors were …
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Figure 3—source data 1
Related to Figure 3A.
Western blot for Dhcr7 in 293T cells transfected with control or individual murine Dhcr7 shRNAs. The blot was re-probed for Erk1/2 as a loading control.
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig3-data1-v2.pdf
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Figure 3—source data 2
Related to Figure 3N.
Western blots of DHCR7 in 293T cells transfected with human control (Con) or human-specific DHCR7 shRNA (sh3) plus human DHCR7-expressing plasmid, analyzed after 24 hr. The blot was re-probed for Erk1/2.
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig3-data2-v2.pdf
Figure 3—figure supplement 1
Rescue of the neurogenesis phenotype in Dhcr7-knockdown mouse cortical precursors by human DHCR7 cDNA expression vector.
Related to Figure 3. Human DHCR7 cDNA expressing vectors transfected murine NPCs with either murine Dhcr7 shRNA vector or control shRNA vectors. The transfected cells were cultured for 3 days, …
Figure 4
7-DHC derived oxysterols lead to similar neurogenic defects as loss of Dhcr7 in murine cortical precursors in vitro.
(A–M) E12.5 cortical precursors were cultured for 2 days in the presence of different concentrations of 7-DHC-derived oxysterols and quantified. (A) Cell were immunostained for Ki67 (green), …
Figure 5
Dhcr7-/- mice display premature neurogenesis and increased activity of the TrkB neurogenic signaling pathway in vivo.
(A) E18.5 cortical sections from Dhcr7+/+ and Dhcr7-/- were immunostained for Tbr1 (red), Ctip2 (green) and counterstained with DAPI (blue). (B and C) Quantifications of the absolute thickness (B) …
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Figure 5—source data 1
Related to Figure 5J.
E15.5 cortices were isolated from Dhcr7+/+ and Dhcr7-/- embryos and analyzed by western blot for phospho-TrkB, phospho-MEK, or phospho-C/EBPβ. Blots were re-probed with antibodies for total GR, TrkB, MEK, C/EBPβ, and GAPDH as loading controls.
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig5-data1-v2.pdf
Figure 6
Dhcr7-/- mice show accelerated cell cycle exiting and depletion of cortical precursor cells in vivo.
(A) Cortical sections from Dhcr7-/- and Dhcr7+/+ embryos labeled by Edu at different developmental stages were immunostained 18 hr later for Edu (green) and Ki67 (Magenta). (B) Quantification of …
Figure 7
Loss of Dhcr7 alters gene expression in cultured SLOS patient-derived neural progenitors.
(A) Hierarchical clustering heatmap of differentially expressed genes shows distinct expression pattern changes in transcript abundance for SLOS mutant NPCs as compared to Controls. Red color …
Figure 8 with 3 supplements
DHCEO activates cortical neurogenesis via activation of glucocorticoid receptor and inhibition of the effect or the formation of DHCEO rescues the neurogenic defects in SLOS NPCs.
(A) Western blot showing increased phospho-GR in E15.5 Dhcr7-/- mouse brain relative to Dhcr7+/+. (B) Image of the docked position of DHCEO (red) and OCDO (blue) in the ligand binding pocket of GR. …
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Figure 8—source data 1
Related to Figure 8A.
Western blot showing increased phospho-GR in E15.5 Dhcr7-/- mouse brain relative to Dhcr7+/+.
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig8-data1-v2.pdf
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Figure 8—source data 2
Related to Figure 8C.
Human neural progenitors were treated with 3.5 μM DHCEO over the indicated time periods. Lysates were probed with phosphor-GR and phosphor-TrkB and re-probed with antibodies for total GR, total TrkB or total ERK as loading controls.
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig8-data2-v2.pdf
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Figure 8—source data 3
Related to Figure 8M.
hNPCs were treated or not treated with MEK/ERK inhibitors, trametinib or PD98059. Western blot of phosphor-MEK. The blots were then re-probed with antibodies for total MEK as loading controls.
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig8-data3-v2.pdf
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Figure 8—source data 4
Related to Figure 8O.
Western blots for TrkB or MEK1/2 in lysate of 293T cells transfected with control or TrkB shRNA or MEK shRNA vector. The blots were re-probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
- https://cdn.elifesciences.org/articles/67141/elife-67141-fig8-data4-v2.pdf
Figure 8—figure supplement 1
Full docking results of DHCEO and OCDO in the binding pocket of human glucocorticoid receptor.
(A) Structures of DHCEO and OCDO. (B) All generated docking positions of DHCEO (red) and ODCO (blue) along with associated scores.
Figure 8—figure supplement 2
Antioxidant and MEK inhibitors rescued the neurogenic phenotype in human and murine NPCs with Dhcr7 mutations.
Related to Figure 8. (A, D) Another SLOS hNPC line (SLOS 5788) and mNPCs from E12.5 Dhcr7-/- and Dhcr7+/+ embryonic cortices were cultured and treated with cholesterol, vitamin E, vitamin C, vitamin …
Figure 8—figure supplement 3
Cholesterol depletion did not disrupt the neurogenesis in human and mouse cultured neural precursors.
Related to Figure 8. (A) Cholesterol levels in human neural precursors (hNPC) treated with vehicle control or different concentrations of β-cyclodextrin determined by LC-MS analysis. (B) Human and …
Additional files
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Supplementary file 1
Retention times and MS/MS transitions for oxysterol internal standards.
Relate to Figure 2 and Figure 2-Figure Supplement 2.
- https://cdn.elifesciences.org/articles/67141/elife-67141-supp1-v2.docx
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Supplementary file 2
Retention times and MS/MS transitions for all oxysterol standards.
Relate to Figure 2 and Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/67141/elife-67141-supp2-v2.docx
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Supplementary file 3
Retention times and MS/MS transitions for sterol internal standards.
Relate to Figure 2 and Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/67141/elife-67141-supp3-v2.docx
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Supplementary file 4
Retention times and MS/MS transitions for sterol standards.
Relate to Figure 2 and Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/67141/elife-67141-supp4-v2.docx
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Supplementary file 5
Ingenuity Pathway Analysis (IPA) reveals “development of the central nervous system” as one of the top 10 enriched Diseases and Biological Functions related to the nervous system.
Relate to Figure 7. DEGs were further analyzed with (IPA) to identify the most enriched biological functions related to the nervous system in SLOS mutant NPCs. The table below shows the top ten enriched terms corresponding to Diseases/Bio-functions along with the p-value and overlapping number of genes in the dataset.
- https://cdn.elifesciences.org/articles/67141/elife-67141-supp5-v2.docx
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Supplementary file 6
Excel file.
List of differentially expressed genes in SLOS hNPCs relative to Control hNPCs. Relate to Figure 7.
- https://cdn.elifesciences.org/articles/67141/elife-67141-supp6-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67141/elife-67141-transrepform1-v2.pdf
