A novel monomeric amyloid β-activated signaling pathway regulates brain development via inhibition of microglia
- Departments of Neurology and Neuroscience, University of Wisconsin-Madison, United States
Figures
Figure 1 with 7 supplements
Deletion of Ric8a using Emx1-Cre results in cortical ectopia due to non-neural deficits.
(a–d) Nissl staining of control (ctrl, a, c) and mutant (mt, b, d) anterior motor (a, b) and posterior somatosensory (c, d) cortex at P0. (e, e’) Laminin (LN, in green) and nuclear …
Figure 1—figure supplement 1
Birth-dating of early- and late-born neurons in Ric8a:Emx1-Cre mutant cortices.
(a–c) BrdU (in red) staining in control (a) and mutant (b) cortices at P5 after administration at E12.5. Quantification is shown in (c). No statistically significant differences were observed …
Figure 1—figure supplement 2
Lamina-specific neuronal markers are normal outside ectopia in Ric8a:Emx1-Cre mutant cortices.
Cux1 (in red) and nuclear (DAPI, in blue) staining of control (a–a”) and mutant (b–b”) cortices at P0 in a region without ectopia. No obvious changes in the expression pattern of Cux1, an upper …
Figure 1—figure supplement 3
Neuronal ectopia in Ric8a:Emx1-Cre mutants result from pial basement membrane breach during embryogenesis.
(a–a”) Laminin (LN, in green), radial glial marker RC2 (in red), and nuclear (DAPI, in blue) staining of control cortices at E16.5. A continuous basement membrane is observed at the pia, where …
Figure 1—figure supplement 4
Basement membrane breaches precede neuronal ectopia in Ric8a:Emx1-Cre mutant cortices.
(a–a”) Laminin (LN, in green) and nuclear (DAPI, in blue) staining of control cortices at E13.5. A continuous basement membrane is observed at the pia, beneath which cells are well organized in the …
Figure 1—figure supplement 5
Signs of basement membrane degradation before breach formation at E12.5.
Laminin (in green) staining of control (a) and Ric8a:Emx1-Cre mutant (b) cortices at E12.5. Increased numbers of laminin-positive debris were observed in mutants (compare insets), even though …
Figure 1—figure supplement 6
Cortical radial glial identity and proliferation are unaffected in Ric8a:Emx1-Cre mutants.
Pax6 (in red) and nuclear (DAPI, in blue) staining of control (a, a’) and mutant (b, b’) cortices at E12.5. Pax6 (in red) and nuclear (DAPI, in blue) staining of control (c, c’) and mutant (d, d’) …
Figure 1—figure supplement 7
Wnt pathway activity is normal in Ric8a:Emx1-Cre mutant cortices.
X-gal staining of BAT-lacZ expression in Ric8a:Emx1-Cre control (a) and mutant (b–d) cortices at E13.5. No obvious differences are observed between controls and three different mutants at this stage.
Figure 2 with 2 supplements
Ric8a deficiency in microglia is responsible for cortical ectopia.
(a) TNFα, IL-1β, and IL-6 secretion (pg/ml) in control and Ric8a:Cx3cr1-Cre mutant microglia following lipopolysaccharide (LPS) stimulation. *p < 0.05; **p < 0.01; ***p < 0.001; n = 6–8 each group. …
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Figure 2—source data 1
Excel files for control and Ric8a mutant microglia ELISA and qRT-PCR analysis.
- https://cdn.elifesciences.org/articles/100446/elife-100446-fig2-data1-v1.zip
Figure 2—figure supplement 1
Emx1-Cre is active in microglia.
TNFα secretion (pg/ml) (a) and basal TNFα and IL-1β mRNA expression (b) in control and Ric8a:Emx1-Cre mutant microglia. *p < 0.05; n = 5–8 each group. (c–c”) The Rosa26 EGFP (green) is induced in …
Figure 2—figure supplement 2
Gαi protein is severely depleted from Ric8a:Emx1-Cre mutant cortices.
Western blot analysis of Gai proteins in E13.5 Ric8a:Emx1-Cre mutant cortices showed that Gαi protein levels were severely reduced (AU, arbitrary units). ***p < 0.001, n = 3 each.
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Figure 2—figure supplement 2—source data 1
Western blot analysis of Gαi levels in E13.5 and P0 brains.
- https://cdn.elifesciences.org/articles/100446/elife-100446-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
Raw scan of western blots of Gαi.
- https://cdn.elifesciences.org/articles/100446/elife-100446-fig2-figsupp2-data2-v1.zip
Figure 3 with 1 supplement
App deficiency results in hypersensitive microglia and cortical ectopia.
(a) TNFα and IL-1β secretion (pg/ml) in cultured control and App:Cx3cr1-Cre mutant microglia following lipopolysaccharide (LPS) stimulation. *p < 0.05; n = 7–9 each group. (b) TNFα, IL-1β, IL-6, and …
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Figure 3—source data 1
Excel files for control and App mutant microglia/macrophage ELISA and qRT-PCR analysis.
- https://cdn.elifesciences.org/articles/100446/elife-100446-fig3-data1-v1.zip
Figure 3—figure supplement 1
Cytokine secretion and transcriptional induction in App:Cx3cr1-Cre mutant microglia.
(a) TNFα and IL-6 secretion (pg/ml) in control and App:Cx3cr1-Cre mutant microglia following overnight lipopolysaccharide (LPS) stimulation. *p < 0.05; **p < 0.01; n = 9–13 each group. (b) TNFα, …
Figure 4 with 1 supplement
Monomeric Aβ40 suppresses microglia via APP and Ric8a.
(a) TNFα, IL-6, IL-1β, and MCP1 secretion (pg/ml) by wildtype microglia following lipopolysaccharide (LPS) stimulation in the absence or presence of Aβ40 (200 or 500 nM). *p < 0.05; ***p < 0.001; n …
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Figure 4—source data 1
Excel files for control and App and Ric8a mutant microglia/macrophage ELISA and qRT-PCR analysis undergoing Aβ40 stimulation.
- https://cdn.elifesciences.org/articles/100446/elife-100446-fig4-data1-v1.zip
Figure 4—figure supplement 1
Effects of monomeric amyloid β (Aβ) on cytokine secretion and transcription in control and mutant microglial lineage cells.
(a) TNFα and IL-6 secretion (pg/ml) in wildtype microglia following lipopolysaccharide (LPS) stimulation in the absence or presence of Aβ40 (50 nM). *p < 0.05; **p < 0.01; n = 25 each group for TNFα …
Figure 5 with 3 supplements
Inhibition of both microglial inflammatory activation and cortical MMP9 activity suppresses basement membrane breach and neuronal ectopia.
Nuclear (DAPI, in gray) staining of untreated (a), anti-inflammatory drug dorsomorphin (DM) (b), Stat3 inhibitor S3I-201 (S3I) (c), and DM/S3I (d) dual treated Ric8a:Emx1-Cre mutant cortices at P0. …
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Figure 5—source data 1
Excel files for Ric8a:Emx1-cre mutant ectopia suppression analysis.
- https://cdn.elifesciences.org/articles/100446/elife-100446-fig5-data1-v1.zip
Figure 5—figure supplement 1
Suppression of astrogliosis in Ric8a:Emx1-Cre mutant cortices by anti-inflammatory drugs, dorsomorphin (DM) and S3I-201 (S3I).
GFAP (in red) and nuclear (DAPI, in blue) staining of neonatal control (a) and mutant cortices without treatment (b) or mutant cortices after dorsomorphin DM (c), S3I-201 S3I (d), or dual DM+S3I (e) …
Figure 5—figure supplement 2
MMP9 in situ and activity in E13.5 Ric8a:Emx1-Cre mutant cortices.
(a) Sections of E13.5 brain wholemount in situ of MMP9 showed a sparse MMP9 expressing cell population resembling microglia (LGE, lateral ganglionic eminence). Tnc5 in situ was performed as control …
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Figure 5—figure supplement 2—source data 1
Whole gel gelatin zymography images of Ric8a:Emx1-Cre control and mutant cortices.
Embryonic lung lysates were used as control for validation of MMP2/9 activity.
- https://cdn.elifesciences.org/articles/100446/elife-100446-fig5-figsupp2-data1-v1.zip
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Figure 5—figure supplement 2—source data 2
Raw scan of zymography data.
- https://cdn.elifesciences.org/articles/100446/elife-100446-fig5-figsupp2-data2-v1.zip
Figure 5—figure supplement 3
Suppression of MMP9 expression in Ric8a:Emx1-Cre mutant cortices by anti-inflammatory drugs, dorsomorphin (DM) and S3I-201 (S3I).
(a) MMP9 (in red) staining in control cortices at E13.5. (b) MMP9 (in red) staining in mutant cortices at E13.5 after DM and S3I dual treatment at E12.5. (c) Quantitative analysis of MMP9 …
