1. Cell Biology
  2. Neuroscience
Download icon

FcγRIIb-SHIP2 axis links Aβ to tau pathology by disrupting phosphoinositide metabolism in Alzheimer's disease model

  1. Tae-In Kam
  2. Hyejin Park
  3. Youngdae Gwon
  4. Sungmin Song
  5. Seo-Hyun Kim
  6. Seo Won Moon
  7. Dong-Gyu Jo
  8. Yong-Keun Jung  Is a corresponding author
  1. Seoul National University, Korea
  2. Institute for Cell Engineering, United States
  3. Johns Hopkins University School of Medicine, United States
  4. Sungkyunkwan University, Korea
Research Article
Cite this article as: eLife 2016;5:e18691 doi: 10.7554/eLife.18691
13 figures and 1 additional file

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 synthetic Aβ1-42 for 24 hr and cell extracts were subjected to western blotting (A). The levels of phosphorylated tau were quantified by densitometry and normalized by total tau (TG5). Values are means ± s.e.m.; *p<0.05, **p<0.005, one-way ANOVA (n = 4) (B). (C) Immunocytochemical analysis of Aβ-induced tau phosphorylation in WT and Fcgr2b KO neurons. (D) Fcgr2b deficiency prevents cell-derived Aβ-induced hyperphosphorylation of tau. Mouse primary hippocampal neurons from WT or Fcgr2b KO embryos were cocultured with CHO or 7PA2 cells for 24 hr (left) and tau phosphorylation was analyzed by western blotting (right). (EF) Rescue of memory impairment in 3xTg-AD/Fcgr2b KO mice. Y-maze (E), novel object recognition (F), and passive avoidance (G) tests were performed in 8–9 month-old WT, Fcgr2b KO, 3xTg-AD, and 3xTg-AD/Fcgr2b KO mice (n = 9–14 mice per group; WT, 5 males and 4 females; KO, 5 males and 4 females; 3xTg-AD, 8 males and 5 females; 3x-Tg-AD/KO, 7 males and 7 females). Data are means ± s.e.m.; *p<0.05, ***p<0.001, unpaired t-test. (H, I) Reduced hyperphosphorylation of tau in 3xTg-AD/Fcgr2b KO mice. The hippocampal lysates of 9 month-old mice were subjected to western blotting (H). The levels of phosphorylated tau were quantified as in (A). Values are means ± s.e.m.; *p<0.05, unpaired t-test (n = 3) (I).

https://doi.org/10.7554/eLife.18691.003
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 Gfap were quantified by real-time PCR in total extract (T), captured neurons (N) or astrocytes (A) (n = 3 mice) (bottom). (B) FcγRIIb protein in neurons. Neurons were isolated from total mouse brain cells (input) using iodixanol density gradient assay. The purified fractions were subjected to western blotting using anti-FcγRIIb (2.4G2), anti-NeuN, and anti-GFAP antibodies.

https://doi.org/10.7554/eLife.18691.004
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 antibody. Compound E (CompE) was used as a negative control and synthetic Aβ1-42 oligomers (200 ng) were used as a loading control. Monomer (mono), dimer (Di) and trimers (Tri) of Aβ1-42 are shown (A). Primary neurons were co-cultured with donor CHO or 7PA2 cells w/wo compound E for 24 hr, and cell lysates were subjected to western blotting (B).

https://doi.org/10.7554/eLife.18691.005
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). Each group mutually did not differ significantly by Student’s t-test and values are means ± s.e.m. (B, C) Age-dependent increase of hyperphosphorylated tau in the hippocampus of 3x Tg-AD mice. The hippocampal lysates of 6 and 20 month-old mice were subjected to western blotting (B). The level of hyperphosphorylated tau detected by PHF1 and CP13 were quantified and normalized by TG5-positive total tau (C). Values are means ± s.e.m.; *p<0.05, **p<0.01, unpaired t-test (n = 3). (D, E) Decrease of hyperphosphorylated tau by Fcgr2b KO in aged 3x Tg-AD mice. The hippocampal lysates of 15 month-old mice were subjected to western blotting (D). The levels of hyperphosphorylated tau (PHF1 and CP13) were quantified and normalized by TG5-positive total tau (E). Values are means ± s.e.m.; *p<0.05, unpaired t-test (n = 3). (F, G) Reduced hyperphosphorylation of tau in hAPP/Fcgr2b KO mice. Hippocampal extracts of 11-month-old WT, Fcgr2b KO, hAPP and hAPP/Fcgr2b KO (hAPP/KO) mice were analyzed by western blotting using the indicated antibodies (D). Levels of the phosphorylated tau were measured by densitometric analysis. Values are means ± s.e.m.; **p<0.005, ***p<0.0005, one-way ANOVA (n = 3) (E).

https://doi.org/10.7554/eLife.18691.006
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 μg/ml purified hFcγRIIb-ED and cell extracts were subjected to western blotting (A). The levels of phosphorylated tau (PHF1, CP13, and AT180) were normalized by total tau (TG5). Values are means ± s.e.m.; *p<0.05, **p<0.005, ***p<0.0005, one-way ANOVA (n = 3) (B). (C, D) Prevention of Aβ1-42-induced tau phosphorylation by anti-FcγRIIb antibody (2.4G2). Mouse primary cortical neurons were pre-incubated with 2.4G2 antibody for 2 hr and treated w/wo 1 μM Aβ1-42 oligomers for 24 hr. Cell extracts were analyzed with western blotting using indicated antibodies (C). Levels of phosphorylated tau were quantified by densitometric measurement. Values are means ± s.e.m.; *p<0.05, **p<0.005, ***p<0.0005, one-way ANOVA (n = 3) (D). (E–G) Suppression of Aβ1-42-induced cognitive deficits by coinjection of anti-FcγRIIb antibody. WT mice (8 weeks old) were i.c.v.-injected with PBS or Aβ1-42 (410 pmol) together w/wo either 2 μg IgG or 2.4G2 antibody. The mice (n = 10 for each group) were analyzed by Y-maze (E; **p<0.005, ***p<0.0005, unpaired t-test), novel object recognition (F; **p<0.005, one-way ANOVA), and passive avoidance (G; *p<0.02, **p<0.005, unpaired t-test) tests as described in the methods. Bars represent means ± s.e.m. (H) Inhibition of i.c.v. Aβ1-42-induced tau phosphorylation by anti-FcγRIIb antibody. Brain extracts of the Aβ- and/or antibody-injected mice were subjected to western blotting.

https://doi.org/10.7554/eLife.18691.007
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 presence or absence of 2.4G2 antibody. Cell lysates were subjected to immunoprecipitation (IP) assay using anti-HA antibody (A) or anti-Aβ (Nu-1) antibody (B). Murine pre-immune (Pre) served as a negative control. The immunoprecipitates were then analyzed with western blotting using anti-Aβ antibody (A) or anti-HA antibody (B). (C) Selectivity of 2.4G2 antibody. Synthetic Aβ1-40, Aβ1-42 oligomers and mouse FcγRIIb-HA-overexpressed HEK293 cell lysates were subjected to western blotting using anti-FcγRIIb (2.4G2) and anti-Aβ antibodies. (D) Total arm entries of the mice i.c.v-injected with oligomeric Aβ alone or together with either immunoglobulin G (IgG) or 2.4G2 were analyzed in Y-maze test (n = 10 mice per group). No significant difference in total arm entries was observed among groups. Data are means ± s.e.m.

https://doi.org/10.7554/eLife.18691.008
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 western blotting using phospho-FcγRIIb and total FcγRIIb antibodies (left). Levels of the phosphorylated FcγRIIb were quantified by densitometric measurement. Values are means ± s.d.; *p<0.05, **p<0.005, two-tailed t-test (n = 3) (right). (B) FcγRIIb is phosphorylated by Aβ1-42 in primary cortical neurons. WT and Fcgr2b KO cortical neurons were incubated with 1 μM Aβ oligomers for 24 hr. Cell lysates were immunoprecipitated using FcγRIIb antibody (2.4G2) and analyzed with western blotting using phospho-tyrosine antibodies. (C) Inhibition of Aβ1-42-induced FcγRIIb phosphorylation by hFcγRIIb-ED protein. SH-SY5Y cells were co-incubated with Aβ1-42 and hFcγRIIb-ED protein. (D) FcγRIIb, not FcγRIIb Y273F-GFP (Y273F), is phosphorylated at Tyr273. (E) FcγRIIb Tyr273 phosphorylation is required for Aβ-induced tau phosphorylation. Fcgr2b KO neurons (DIV 8) were transfected with the indicated constructs, followed by incubation with 1 μM Aβ1-42. Cell extracts were subjected to western blotting (left). The signals on the blot were quantified and bar graph represents phospho-tau levels normalized by TG5 (right). All data shown are means ± s.e.m.; *p<0.05, **p<0.005, one-way ANOVA. (F) FcγRIIb is phosphorylated at Tyr273 in AD brains. Hippocampal homogenates from normal, MCI (Braak III), and AD patients (Braak V/VI) were analyzed with western blotting (left). Levels of phosphorylated FcγRIIb and total FcγRIIb were quantified and values are means ± s.e.m.; **p<0.01, two-tailed t-test (right).

https://doi.org/10.7554/eLife.18691.009
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 subjected to western blotting using PHF1, Tau5, p-GSK3β, GSK3β, p35/p25, FcγRIIb (2.4G2) and β-actin antibodies (A). The signals of p-GSK3β, GSK3β and p25 in (A) were measured by densitometric analysis. Values are means ± s.e.m. (n = 3). *p<0.05, one-way ANOVA (B). (C-F) Prevention of FcγRIIb-mediated tau hyperphosphorylation by GSK3b inhibitor. SH-SY5Y cells (C, D) or primary cortical neurons (E, F) were transfected with vehicle or pFCGR2B-flag alone (E, F) or together with GFP-tau (C, D) for 36 hr and then exposed to the indicated doses of SB-415286 (SB) (C, E) or roscovitine (Rosco) (D, F) for 12 hr. Cell extracts were prepared and subjected to western blot analysis using the indicated antibodies.

https://doi.org/10.7554/eLife.18691.010
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 domain, Cyto; cytosolic domain (left). HT22 cells were transiently transfected with pEGFP, FcγRIIb-GFP (WT), FcγRIIb ITIM-deleted form (1-270; ΔITIM), FcγRIIb cytosolic domain-deleted form (1-240; ΔCyto) or FcγRIIb phosphorylation-defective mutant in ITIM (Y273F). GFP-positive cells showing condensed and fragmented nuclei after staining with Hoechst 33258 were counted as apoptotic cells under a fluorescence microscope. At least 900 cells in 3–4 random spots per sample per condition were counted. Values are means ± s.d.; n = 3 (right). (B) A FcγRIIb Y273F mutant prevents Aβ neurotoxicity. SH-SY5Y cells were transfected with WT or FcγRIIb Y273F mutant, and then incubated w/wo oligomeric Aβ1-42. Bars depict the incidence of cell death. Values are means ± s.d; n = 3. *,#p<0.05, **,##p<0.005, unpaired t-test, compared to cells transfected with pEGFP (Mock) and treated with PBS (*, **) or synthetic Aβ (#, ##), respectively. N.S, not significant.

https://doi.org/10.7554/eLife.18691.011
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 were subjected to immunoprecipitation analysis using anti-His (A) and anti-GFP (B) antibody, respectively. Whole cell lysates (WCL) and the immunoprecipitates were probed by western blotting using anti-GFP and anti-His antibodies. (C, D) Regulated interaction between SHIP2 and FcγRIIb2 in response to Aβ1-42. SH-SY5Y cells were left untreated or incubated with 1 μM Aβ1-42 oligomers for 24 hr and cell extracts were immunoprecipitated using anti-FcγRIIb antibody (C) or anti-SHIP2 antibody (D). (E) Aβ induces colocalization of FcγRIIb and SHIP2 on the plasma membrane. SH-SY5Y cells were incubated with 1 μM Aβ1-42 for 24 hr and then subjected to immunocytochemical analysis using anti-FcγRIIb and anti-SHIP2 antibodies. Hoechst dye was used for nuclear staining. (F) Increased targeting of SHIP2 to the plasma membrane by Aβ1-42. SH-SY5Y cells were treated with 1 μM Aβ1-42 for 24 hr and then subjected to subcellular fractionation assay to separate the plasma membrane from the cytosol. The fractions were analyzed by western blotting. The α-tubulin antibody was used as a marker for the cytosolic fraction (left). The relative expression of SHIP2 at each fraction was quantified by densitometric analysis (right). Values are means ± s.d.; *p<0.05, two-tailed t-test.

https://doi.org/10.7554/eLife.18691.012
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 indicate means ± s.e.m.; N.S. not significant, one-way ANOVA (B).

https://doi.org/10.7554/eLife.18691.013
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)Plevels 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 and analyzed by PLO assay using purified TAPP1-PH and GRP1-PH proteins which bind to PtdIns(3,4)P2 and PtdIns(3,4,5)P3, respectively (A) or analyzed by ELISA to quantify PtdIns levels (C). The signals on the blots in (A) were quantified by densitometric analysis (B). All data shown are means ± s.d.; **p<0.005, ***p<0.0005, unpaired t-test. (D) Aβ1-42 increase PtdIns(3,4)P2 levels at the plasma membrane through FcγRIIb. SH-SY5Y/pSuper and SH-SY5Y/shFCGR2B stable cells were transfected with the GFP-PHTAPP1 probe and stimulated with 1 μM Aβ1-42 oligomers for 24 hr. The fluorescence of GFP-PHTAPP1 was observed by confocal microscopy (top). The fluorescence intensity of the GFP-PHTAPP1 probe in the plasma membrane (two external peaks) was quantified after Aβ1-42 treatment (bottom). (E) Intracellular delivery of PtdIns(3,4)P2 induces tau hyperphosphorylation. Primary cortical neurons were incubated with the indicated concentrations of PtdIns(3,4)P2 w/wo carriers for 24 hr and cell lysates were subjected to western blotting.

https://doi.org/10.7554/eLife.18691.014
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 onto nitrocellulose membranes and the membrane was then incubated with purified His-TAPP1-PH protein. His-fusion proteins bound to the phosphoinositides were detected by western blotting using His antibody (left). Primary cortical neurons were incubated with PBS or Aβ oligomers for 24 hr and cell extracts were analyzed by a PLO assay using purified TAPP1-PH protein (right). (B) Requirement of FcγRIIb and SHIP2 for Aβ-induced PtdIns(3,4)P2 dysregulation. SH-SY5Y/pSuper, SH-SY5Y/shFCGR2B and SH-SY5Y/shINPPL1 knockdown cells were incubated with Aβ1-42 oligomers for 24 hr, and TAPP1-PH-bound PtdIns(3,4)P2 or GRP1-PH-bound PtdIns(3,4,5)Pwas then analyzed by a PLO assay. (C) No difference between SH-SY5Y/pSuper and SH-SY5Y/shFCGR2B cells in H2O2-induced localization of GFP-PHTAPP1 to the plasma membrane. SH-SY5Y/pSuper and SH-SY5Y/shFCGR2B cells were transfected with GFP-PHTAPP1 for 24 hr and treated with 300 μM H2O2 for 1 hr.

https://doi.org/10.7554/eLife.18691.015
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 were subjected to western blotting. (B) Alleviating ER stress reduces PtdIns(3,4)P2-evoked GSK3β activation and tau hyperphosphorylation. Primary cortical neurons (DIV 10) were preincubated with 3 mM 4-phenylbutyric acid (4-PBA) or 75 μM Salubrinal for 2 hr and treated with 10 μM PtdIns(3,4)P2 for 24 hr. Cell lysates were subjected to western blotting. (C) Suppression of Aβ-induced ER stress and GSK3β activation by SHIP2 inhibitor. Primary cortical neurons (DIV 10) were preincubated with 10 μM AS1949490 (AS) for 2 hr and treated with 1 μM Aβ1-42 oligomers for 24 hr. Cell lysates were subjected to western blotting. (D) Suppression of Aβ-induced ER stress and GSK3β activation by Inppl1 knockdown. Primary cortical neurons (DIV 10) were infected with control or Inppl1 siRNA-containing lentivirus (LV-siInppl1) for 3 days, and then incubated with 1 μM Aβ1-42 oligomers for 24 hr. Cell lysates were subjected to western blotting.

https://doi.org/10.7554/eLife.18691.016
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, cells were incubated with 1 μM Aβ1-42 oligomers for 24 hr and Aβ-induced tau phosphorylation was analyzed by western blotting. (B–D) Prevention of memory impairments and tau hyperphosphorylation by Inppl1 knockdown in 3xTg-AD mice. Time schedule of the memory experiments (top). LV-Com or LV-siInppl1 was injected into the dentate gyrus of 7–8 month-old mice. Beginning 20 days after the injection, mice were subjected to Y-maze (B) and novel object recognition (C) tests (n = 7–10 mice per group). *p<0.05, **p<0.005, ***p<0.0005, one-way ANOVA. The hippocampal lysates were subjected to western blotting (D). (E) Suppression of Aβ-induced tau phosphorylation by a SHIP2 inhibitor. Primary cortical neurons were incubated with AS1949490 for 2 hr and then with 1 μM Aβ1-42 for 24 hr. Cell extracts were analyzed with western blotting (top). The signals on the blots were quantified. *p<0.01, **p<0.005, unpaired t-test (bottom). (F–H) SHIP2 inhibitor prevents Aβ-induced memory deficits. The 2-month-old WT mice were injected with Aβ1-42 alone or together with 10 μg AS1949490 (n = 10 per groups). One day later, mice were analyzed with Y-maze (F), novel object recognition (G), and passive avoidance (H) tests. **p<0.005, ***p<0.0005, one-way ANOVA. (I) Suppression of Aβ-induced tau phosphorylation in vivo by SHIP2 inhibitor. The hippocampal extracts were subjected to western blotting. All data are means ± s.e.m. (n = 3).

https://doi.org/10.7554/eLife.18691.017
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 untreated or incubated with 5 μM Aβ1-42 for 24 hr. Cell extracts were subjected to western blotting. (B, C) Requirement of SHIP2 in Aβ- and FcγRIIb-induced cell death. SH-SY5Y/pSuper and SH-SY5Y/shINPPL1 cells were treated with oligomeric Aβ1-42 for 48 hr (B) or transfected with pFcγRIIb for 36 hr (C), and cell death was then examined. Values are means ± s.d.; n = 3. *p<0.05, ***p<0.0005, two-tailed t-test. (D) Increase of tau phosphorylation by ectopic expression of SHIP2. SH-SY5Y cells were transiently transfected with pGFP-tau alone or together with pSHIP2 for 36 hr. Cell lysates were subjected to western blotting. (E) Requirement of inositol phosphatase activity of SHIP2 in the activation of AKT-GSK3β pathway. SH-SY5Y cells were transfected with SHIP2 WT or D608A catalytically inactive mutant for 36 hr. Cell extracts were analyzed by western blotting.

https://doi.org/10.7554/eLife.18691.018
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 infected by LV-empty or LV-siInppl1 for 4 days and knockdown of SHIP2 expression was assessed by western blotting.

https://doi.org/10.7554/eLife.18691.019
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 viability was determined using Calcein-AM assay. Data are means ± s.d. (n = 3); **p<0.001, One-way ANOVA. (B) Total arm entries of AS1949490-injected mice were analyzed in Y-maze test (n = 10 per groups). Data are means ± s.e.m.

https://doi.org/10.7554/eLife.18691.020
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 with pSUPER-neo (Mock) or pLyn shRNA (shLyn) for 48 h and then incubated with PBS or 2 µM Aβ1- 42 for additional 12 h. Cell lysates were immunoblotted using the indicated antibodies. (C) HT22 cells were incubated with DMSO, 0.5 µM Lyn inhibitor or Scramble, and then transfected with pEGFP-N1 (Mock) or pFcγRIIb-EGFP (FcγRIIb) for 36 h. Cells showing fragmented nuclei after staining by EtHD were considered as dead cells. Values are means ± s.d. (n = 3). ***P<0.0005, **P<0.005, two-tailed t-test. (D) SH-SY5Y cells were pre-treated with the indicated concentrations (µM) of Lyn inhibitor or scramble for 2 h, and then transfected with pFcγRIIb-EGFP for additional 24 h. Cell lysates were immunoblotted. (E) Primary cortical neurons at DIV 5 were pre-treated with DMSO or the indicated concentrations of small molecules (Lyn inhibitor and scramble) for 2 h, and then incubated with 5 µM Aβ1-42 for 48 h. Values are means ± s.d. (n = 3). #P<0.005 versus PBS with DMSO-treated sample. *P<0.05, **P<0.005 versus Aβ1-42 with DMSO-treated sample. two-tailed t-test.

https://doi.org/10.7554/eLife.18691.022
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 neurons. (F) Increase in Fcgr2b mRNA by Aβ1-42 in primary cortical neurons. RT-PCR analysis was performed with synthetic primers.

https://doi.org/10.7554/eLife.18691.023
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 primary cortical neurons.

https://doi.org/10.7554/eLife.18691.024
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 showing neuronal expression of FcγRIIb-DN in the transgenic mice. n.s. non-specific signals. (C) Genomic PCR analysis showing generation of 3xTg-AD/FcγRIIb DN double transgenic mice. (D) Behavior tests showing memory rescue in the 3xTg-AD mice by FcγRIIb DN expression. (left) Y-maze test, (middle) noble object recognition test, (right) passive avoidance test. (n = 9-11 male mice per group). Values are means ± s.e.m.; *P<0.05, **P<0.01, ***P<0.005, unpaired t-test.

https://doi.org/10.7554/eLife.18691.025
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 section of brain at P56 were displayed. Probes that span from exon 1 to exon 7 of Fcgr2b transcript were used. Arrows indicate the positive signals located in either pyramidal cells in CA1 and CA3 or granule cells in dentate gyrus (DG). Bar, 200 µm.

https://doi.org/10.7554/eLife.18691.026
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) Quantification of Iba1 immunoreactivity in the cortex (B) and hippocampus (C) of 3x Tg-AD and 3x Tg-AD/Fcgr2b KO mice. Values are means ± s.e.m. (n = 8). *P<0.05 compared with 3x Tg-AD mice. (D) Expression of IL1β, iNOS, and TNFα were diminished in 3x Tg-AD/Fcgr2b KO mice. Total RNAs were extracted from cortical lysates from 6 month-old 3x Tg-AD and 3x Tg-AD/Fcgr2b KO mice and were analyzed by quantitative RT-PCR. Values are means ± s.e.m. (n = 3). *P<0.05.

https://doi.org/10.7554/eLife.18691.027
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 phosphorylation-defective mutant in ITIM (Y273F) for 24 h. Cell viability were determined by using EZ-CYTOXTM cell viability kit Values are means ± s.e.m. (n = 3). **P<0.01, N.S., not significant, compared with Mock control.

https://doi.org/10.7554/eLife.18691.028

Additional files

Supplementary file 1

Primer sequences for cloning, shRNA and qPCR.

https://doi.org/10.7554/eLife.18691.021

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)