FcγRIIb-SHIP2 axis links Aβ to tau pathology by disrupting phosphoinositide metabolism in Alzheimer's disease model
- Seoul National University, Korea
- Institute for Cell Engineering, United States
- Johns Hopkins University School of Medicine, United States
- Sungkyunkwan University, Korea
Figures
Figure 1 with 3 supplements
Fcgr2b deficiency prevents tau hyperphosphorylation and memory deficits in 3xTg mice.
(A, B) Fcgr2b KO neurons are resistant to Aβ-induced tau phosphorylation. Mouse primary cortical neurons from wild-type (WT) or Fcgr2b KO embryos (DIV 8) were incubated with oligomeric forms of 1 μM …
Figure 1—figure supplement 1
Neuronal expression of FcγRIIb in the mouse brain.
(A) Fcgr2b mRNA level in captured neurons. NeuN- or GFAP-positive cells were isolated from wild-type mouse brains by laser capture microdissection (top). Relative mRNA levels of Fcgr2b, Neun, and Gfa…
Figure 1—figure supplement 2
FcγRIIb is required for cell-derived Aβ oligomer-induced tau phosphorylation in the primary neurons.
(A, B) Induction of tau phosphorylation in primary neurons by naturally secreted Aβ. The cell-derived Aβ in 7PA2-CHO cells were detected by immunoprecipitation and western blot analysis using Aβ …
Figure 1—figure supplement 3
FcγRIIb is also required for Aβ-induced tau phosphorylation in the hippocampus of aged 3xTg-AD and hAPP (J20) mouse lines.
(A) Aβ levels in the hippocampus of 3xTg-AD and 3xTg-AD/Fcgr2b KO mice. Levels of Aβ1-40 and Aβ1-42 in the hippocampus of 9-month-old 3xTg-AD or 3xTg-AD/KO mice were determined by ELISA (n = 3). …
Figure 2 with 1 supplement
Inhibition of Aβ1-42-FcγRIIb interaction blocks Aβ-induced tau phosphorylation and memory impairment.
(A, B) Inhibition of Aβ1-42-induced tau phosphorylation by the addition of purified hFcγRIIb-ED protein. Mouse primary cortical neurons were incubated for 24 hr with 1 μM synthetic Aβ1-42 w/wo 50 …
Figure 2—figure supplement 1
FcγRIIb-antagonizing antibody, 2.4G2, inhibits the interaction of Aβ1-42 with FcγRIIb.
(A, B) Inhibition of the interaction between Aβ and FcγRIIb by 2.4G2 antibody. SH-SY5Y cells that stably express hFcγRIIb-HA were left untreated or incubated with 1 μM Aβ1-42 for 1 hr in the …
Figure 3 with 2 supplements
FcγRIIb Tyr273 phosphorylation is found in AD brains and mediates Aβ-induced tau phosphorylation.
(A) Human FcγRIIb is phosphorylated at Tyr273 by Aβ1-42. Wild-type and FCGR2B knockdown SH-SY5Y cells (SH-SY5Y/shFCGR2B) were incubated with 1 μM Aβ1-42 oligomers and cell extracts were subjected to …
Figure 3—figure supplement 1
FcγRIIb-mediated tau phosphorylation is dependent on GSK3β, not CDK5.
(A, B) Inhibition of Aβ-induced GSK3β activation in Fcgr2b KO neurons. Primary cortical neurons from WT or Fcgr2b KO embryos (DIV 8) were incubated with 5 μM Aβ1-42 for 24 hr. Cell extracts were …
Figure 3—figure supplement 2
Phosphorylation of FcγRIIb Tyr273 is required for Aβ neurotoxicity.
(A) Association of the neurotoxic activity of FcγRIIb with ITIM. A schematic diagram showing the structure of FcγRIIb protein. SS; signal sequence, EX; extracellular domain, TM; transmembrane …
Figure 4 with 1 supplement
SHIP2 is recruited and binds to phosphorylated FcγRIIb in response to Aβ.
(A, B) Interaction between phosphorylated FcγRIIb and SHIP2. SH-SY5Y cells were co-transfected with pSHIP2-His and either pFcγRIIb WT-GFP (WT) or pFcγRIIb Y273F-GFP (YF) for 36 hr and cell extracts …
Figure 4—figure supplement 1
Expression of SHIP2 in human brain.
(A, B) Hippocampal extracts from normal, MCI (Braak III), and AD patients (Braak V/VI) were analyzed with western blotting (A). Levels of SHIP2 were quantified by densitometric analysis and bars …
Figure 5 with 2 supplements
FcγRIIb-SHIP2 axis deregulates PtdIns(3,4)P2 metabolism for tau phosphorylation.
(A–C) Aβ1-42 increase PtdIns(3,4)P2 levels through FcγRIIb. Primary cortical neurons from WT and Fcgr2b KO embryos were incubated w/wo 1 μM Aβ1-42 oligomers for 24 hr. Total lipids were extracted …
Figure 5—figure supplement 1
The FcγRIIb-SHIP2 axis is required for Aβ-induced PtdIns(3,4)P2 dysregulation for tau phosphorylation.
(A) Aβ oligomers increase TAPP1-PH-bound PtdIns(3,4)P2 levels in primary cortical neurons. Serial dilutions (500, 250, 125, 62.5, 32, and 16 pmol) of the indicated phosphoinositides were spotted …
Figure 5—figure supplement 2
ER stress links SHIP2 to GSK3β for tau phosphorylation.
(A) Cellular delivery of PtdIns(3,4)P2 triggers ER stress and GSK3β activation. Primary cortical neurons (DIV 7) were incubated with 10 μM phosphoinositides with carriers for 24 hr. Cell lysates …
Figure 6 with 3 supplements
Lentiviral or pharmacological inhibition of SHIP2 prevents Aβ-mediated memory impairments and tau phosphorylation in vivo.
(A) Suppression of Aβ-induced tau phosphorylation by Inppl1 knockdown. Primary cortical neurons were infected with control (LV-Con) or Inppl1 siRNA-containing lentivirus (LV-si Inppl1). On day 3, …
Figure 6—figure supplement 1
SHIP2 is required for Aβ-induced tau phosphorylation and neurotoxicity.
(A) Inhibition of Aβ-induced tau phosphorylation by INPPL1 knockdown in SH-SY5Y cells. SH-SY5Y/pSuper and SH-SY5Y/shINPPL1 cells were infected with GFP-tau adenovirus for 36 hr and then left …
Figure 6—figure supplement 2
Knockdown of SHIP2 expression with siRNA-carrying lentivirus
(A) HT22 cells were infected by lentivirus (LV)-empty or LV-siInppl1 with MOI 2 or 5. After 5 days, SHIP2 expression was analyzed by western blotting. (B) Primary cortical neurons (DIV 8) were …
Figure 6—figure supplement 3
A SHIP2 inhibitor prevents Aβ-induced neurotoxicity.
(A) Inhibition of Aβ-induced cell death in neurons by SHIP2 inhibitor. Primary cortical neurons were preincubated with 10 μM AS1949490 for 2 hr and further treated with Aβ1-42 for 48 hr. Cell …
Author response image 1
Lyn is required for FcγRIIb phosphorylation to mediate Ap neurotoxicity.
(A) Primary cortical neurons (DIV 5) and SH-SY5Y cells were treated with PBS or 5 µM Aβ1-42 for 24 h. Cell lysates were prepared and subjected to western blotting. (B) SH-SY5Y cells were transfected …
Author response image 2
Expression of FcγRIIb protein and mRNA in the purified neurons.
(C, D) Detection of FcγRIIb protein in NeuN- positive neurons of normal and AD patients by immunohistochemistry. (E) Detection of FcγRIIb protein by western blotting in the primary cultured cortical …
Author response image 3
Detection of neuron-enriched FcetIIb isoform, FcγRIIb2.
(A) RT-PCR analysis showing Fcgr2b mRNA in the purified neurons of WT and Fcgr2b KO brain and other tissues. (B) Increase of Fcgr2b2 mRNA by oligomeric Aβ1-42, but not by monomeric Aβ1-42, in …
Author response image 4
Neuron-specific effect of FcγRIIb-dominant negative mutant on memory function in the transgenic mice.
(A) Construction of CamKIIα promoter-FcγRIIb deletion mutant lacking its cytoplasmic domain (∆232-300) (FcγRIIb DN). Arrows indicate PCR primers for genotyping of the mice. (B) Western blotting …
Author response image 5
Expression pattern of Fcgr2b in mouse hippocampus from Allen Brain Atlas.
Website: © 2015 Allen Institute for Brain Science. Allen Brain Atlas [Internet]. Available from: http://www.brain-map.org. In situ hybridization (A) and expression image (B) of Fcgr2b in sagittal …
Author response image 6
Microglial activation is suppressed by Fcgr2b KO in 3x Tg-AD mice.
(A) Immunohistochemical analysis of Iba1 levels in the cortex and hippocampus of 7 month-old 3x Tg-AD and 3x Tg-AD/Fcgr2b KO mice. Magnifying power 200x. White scale bar, 20 µm. (B, C) …
Author response image 7
Association of the neurotoxic activity of FcγRIIb with ITIM.
HT22 cells were transiently transfected with pEGFP (Mock), FcγRIIb-GFP (WT), FcγRIIb ITIM-deleted form (1-270; ΔITIM), FcγRIIb cytosolic domain-deleted form (1-240; ΔCyto), or FcγRIIb …
Additional files
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Supplementary file 1
Primer sequences for cloning, shRNA and qPCR.
- https://doi.org/10.7554/eLife.18691.021
