Fig 1.
Microglia response to LPS-induced inflammation is hampered in 3 month old APPKO mice.
(A-H) Representative brain sections immuno-stained with anti-Iba1 in hippocampus of 3 month old mice (A-D), higher magnification shown in (E-H). Iba-1 expression in microglial cells was increased 3 days after LPS injection (C,D,G,H) in both APPKO and wild type C57BL6 mice. However, this increase in microglial Iba1 immuno-staining was blunted in APPKO mice (D, H) compared to APP sufficient C57BL6 wild type mice (C,G). Quantitative RT-PCR for Iba-1 mRNA levels shows significant reductions in Iba-1 transcripts in APPKO mice 3 days after LPS injection (I). Detailed cell count and morphological analysis show that the number of Iba-1 positive microglia cells in the hippocampus were less in APPKO mice (J), the microglial cells present with shorter processes in APPKO mice (K) and the microglial cell body area was smaller in APPKO mice (L) in response to LPS stimulation. * p<0.05, *** p<0.001 APPKO vs C57BL6.
Fig 2.
Astrocytes response to LPS-induced inflammation is hampered in 3 month old APPKO mice.
(A-H) Representative brain sections immuno-stained with anti-GFAP in hippocampus of 3 month old mice (A-D), higher magnification shown in (E-H). GFAP expression in astrocytic cells was increased 3 days after LPS injection (C,D,G,H) in both APPKO and wild type C57BL6 mice. However, this increase in GFAP immuno-staining was blunted in APPKO mice (D, H) compared to APP sufficient C57BL6 wild type mice (C,G). Quantitative RT-PCR for GFAP mRNA levels shows significant reductions in GFAP transcripts in APPKO mice 3 days after LPS injection (I). Detailed cell count and morphological analysis show that the numbers of GFAP positive astrocytes in the hippocampus were less in APPKO mice compared to BL6 mice (J), astrocytic cells present with shorter processes in APPKO mice (K) and the cell body area is smaller in APPKO mice (L) in response to LPS stimulation. *** p<0.001 APPKO vs C57BL6.
Fig 3.
Microglia response to LPS-induced inflammation is hampered in 9 months old APPKO mice.
(A-H) Representative brain sections immuno-stained with anti-Iba-1 in hippocampus of 9 month old mice (A-D), higher magnification shown in (E-H). Iba-1 expression in microglial cells was increased 3 days after LPS injection (C,D,G,H) in both APPKO and wild type C57BL6 mice. However, this increase in microglial Iba1 immuno-staining was blunted in APPKO mice (D, H) compared to APP sufficient C57BL6 wild type mice (C,G). qPCR for Iba-1 mRNA levels show reductions in Iba-1 transcripts in APPKO mice 3 days after LPS injection (I). Detailed cell count and morphological analysis showed that the numbers of Iba-1 positive microglia in the hippocampus were less in APPKO mice (J), the microglial cells present with shorter processes in APPKO mice (K) and the microglial cell body area was smaller in APPKO mice (L) in response to LPS stimulation. * p<0.05 APPKO vs C57BL6.
Fig 4.
Astrocytes response to LPS-induced inflammation in 9 months old APPKO mice.
(A-H) Representative brain sections immuno-stained with anti-GFAP in hippocampus of 9 month old mice (A-D), higher magnification shown in (E-H). GFAP expression in astrocytes was increased 3 days after LPS injection (C,D,G,H) in both APPKO and wild type C57BL6 mice. (I) qPCR analysis showed increased expression of GFAP mRNA levels in both APPKO mice and BL6 mice following LPS stimulation, however, there were no significant differences in GFAP mRNA levels between APPKO mice compared to BL6 mice. Detailed cell count and morphological analysis showed that the numbers of GFAP positive cells in the hippocampus were comparable in APPKO mice and BL6 (J), there were no significant differences in cell processes length (K) nor cell body area (L) in APPKO mice compared to BL6 in response to LPS stimulation.
Fig 5.
Microglial markers profile in APPKO mice after LPS treatment
Quantitative RT-PCR analysis of hippocampal mRNA levels from 3-month old (A) and 9 months old mice (B) showed altered expression pattern of several microglia markers in APPKO mice compared to C57BL6 wild type mice in response to hippocampal LPS injection at 3 days after surgery. Data is represented as fold change over untreated control mice. *p<0.05, ** p<0.01, *** p<0.001 (C57BL6 vs APPKO).
Fig 6.
Reduced inflammatory cytokine levels in APPKO mice after LPS treatment.
Quantitative RT-PCR analysis of hippocampal mRNA levels from 3 months old (A) and 9 months old (B) mice showed reduced expression pattern of several inflammatory cytokines (TNF-α, IL-1β, Il-10 and TGFβ) in response to LPS injection. Data is represented as fold change over untreated control mice. *p<0.05, ** p<0.01, *** p<0.001 (C57BL6 vs APPKO).
Fig 7.
Changes in neuronal markers in APPKO mice after LPS treatment.
Quantitative RT-PCR analysis of hippocampal mRNA levels of neuronal markers (synaptophysin, PSD-95 and BDNF) in 3 months old (A) and 9-months old (B) APPKO mice compared to C57BL6 wild type mice in response to LPS injection. Data is represented as fold change over untreated control mice. *p<0.05, ** p<0.01, *** p<0.001 (C57BL6 vs APPKO).
Fig 8.
Lack of APP reduces the microglial response in vitro.
(A) Primary microglia cells isolated from APPKO mice, challenged with LPS for 4 hours expressed lower mRNA levels of Iba-1, IL-6 and TNF-α compared to BL6 cells. (B) Representative anti-APP CT20 immunoblot confirmed the lack of APP protein both in primary microglial cells and brain lysates isolated from APPKO mice. (C) qPCR for APP mRNA showed no significant differences following LPS treatment in vitro and in vivo. ** p-value<0.01.