Research ArticleAlzheimer’s Disease

Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice

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Science Translational Medicine  31 May 2017:
Vol. 9, Issue 392, eaaf6295
DOI: 10.1126/scitranslmed.aaf6295
  • Fig. 1. APP/PS1;C3 KO mice show improved cognitive flexibility (reversal) compared to APP/PS1 mice at 16 months of age.

    (A) Percent of mice that reached criterion (≥80% correct choices on each individual day) in the water T-maze test. Compared to WT mice, APP/PS1 mice were impaired in acquisition (day 2) and reversal learning and memory (days 4 and 5) (*P < 0.05, **P < 0.01). APP/PS1;C3 KO mice performed significantly better than did APP/PS1 mice (##P < 0.01), but similar to WT and C3 KO mice, in the reversal test on days 4 and 5, suggesting better cognitive flexibility in APP/PS1;C3 KO mice compared to APP/PS1 mice. (B) In total, fewer APP/PS1 mice reached the reversal criterion (≥80% correct choices over two consecutive days) (*P < 0.05), whereas the percent of WT, C3 KO, and APP/PS1;C3 KO mice that reached criterion in the reversal test was higher compared to APP/PS1 mice (***P < 0.001), indicating that C3 deficiency in APP/PS1 mice had both age-dependent and AD-related effects (WT, n = 13; APP/PS1, n = 11; APP/PS1;C3 KO, n = 10; C3 KO, n = 11). Tests were assessed using one-way analysis of variance (ANOVA) followed by Fisher’s protected least significant difference post hoc test.

  • Fig. 2. Increased Aβ plaque load in 16-month-old APP/PS1;C3 KO mice.

    (A and B) Quantification of Aβx–42, Aβx–40, and Aβ1–x immunoreactivities within a designated region of interest (ROI) revealed increased plaque burden in the cortex (A) and hippocampus (B) of 16-month-old APP/PS1;C3 KO mice compared to APP/PS1 mice (*P < 0.05, **P < 0.01 versus APP/PS1 mice, independent unpaired t test per Aβ species; n = 9 mice; six equidistant planes, 150 μm apart). (C) Aβx–42–immunoreactive and thioflavin S–positive plaques were higher in the hippocampus of C3-deficient APP/PS1 mice versus APP/PS1 mice. White circles indicate large plaques (>50 μm); black arrows indicate medium-sized plaques (>20 but <50 μm). Scale bars, 50 μm. (D) Quantification of small, medium, and large hippocampal plaques confirmed an increased plaque load in APP/PS1;C3 KO mice, especially for large plaques (*P < 0.05, **P < 0.01, independent unpaired t tests per plaque size category; n = 10). (E) Quantification of thioflavin S (Thio S) in hippocampus confirmed an increase in fibrillar plaques in APP/PS1;C3 KO mice (*P < 0.05 versus APP/PS1 mice, unpaired t test; n = 6; three equidistant planes 300, μm apart). (F) Increased insoluble cerebral Aβx–40 and Aβx–38 were found in APP/PS1;C3 KO mice compared with APP/PS1 mice (*P < 0.05, independent unpaired t tests per Aβ species; n = 8).

  • Fig. 3. Morphological changes associated with glial activation were reduced in 16-month-old APP/PS1;C3 KO mice.

    (A) Iba-1– and CD68-positive immunostaining showed less activation of microglia and macrophages (that is, smaller cells with thinner processes) and less clustering of GFAP-positive astrocytes in the hippocampal CA3 region of 16-month-old APP/PS1;C3 KO mice compared to APP/PS1 mice. Scale bar, 50 μm. (B to D) Quantification of Iba-1, CD68, and GFAP immunostaining in hippocampal CA3, CA1, and dentate gyrus (DG) showed reduced glial immunoreactivity (IR) in APP/PS1;C3 KO mice compared to APP/PS1 mice (*P < 0.05, **P < 0.01, independent unpaired t tests per region; n = 6 to 8; three equidistant planes, 300 μm apart). (E) High-resolution confocal images of Aβ plaques immunostained with 6E10 antibody show microglia/macrophages (immunoreactive for Iba-1) and phagocytic cells (immunoreactive for CD68) in the hippocampal CA3 region. These findings suggested reduced phagocytosis in APP/PS1;C3 KO mice compared to APP/PS1 mice. Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (F) Iba-1– and CD68-positive immunofluorescence intensities were lower in APP/PS1;C3 KO mice compared to APP/PS1 mice (**P < 0.01, independent unpaired t tests per marker; n = 5). (G and H) Stereological counts of Iba-1–immunoreactive cells (G) and GFAP-immunoreactive cells (H) counterstained with 3,3′-diaminobenzidine were performed in hippocampal CA3, CA1, and dentate gyrus tissue. The number of Iba-1–positive microglia/macrophages was increased in CA3 and dentate gyrus in APP/PS1 and APP/PS1;C3 KO mice versus WT and C3 KO mice (G). The number of GFAP-positive astrocytes was increased only in the hippocampal CA3 region (H); no differences were observed in glial cell numbers between APP/PS1 and APP/PS1;C3 KO mice (**P < 0.01, one-way ANOVA with Bonferroni post hoc test per region; n = 6 to 8; three equidistant planes, 300 μm apart).

  • Fig. 4. Plaque-associated microglia and astrocytes and brain cytokines were altered in APP/PS1;C3 KO mice compared to APP/PS1 mice.

    (A, B, G, and H) High-resolution confocal images of Iba-1 (red)/6E10 (Aβ antibody; green)/DAPI (blue) or GFAP (red)/6E10 (green)/DAPI (blue) in APP/PS1 and APP/PS1;C3 KO mice. The inner ring indicates the proximal region of a plaque (that is, the center), whereas the outer ring indicates the distal region. Scale bars, 10 μm. (C, D, I, and J) Immunofluorescence intensities of Iba-1 and GFAP were lower in the Aβ plaque proximal area (C and I) and higher in the Aβ plaque distal area (D and J) in APP/PS1;C3 KO mice compared to APP/PS1 mice (*P < 0.05, **P < 0.01, unpaired t test; n = 6). (E, F, K, and L) The number of Iba-1– and GFAP-positive cells was reduced in the proximal plaque area in APP/PS1;C3 KO mice compared to APP/PS1 mice (E and K) (*P < 0.05) and increased in the distal plaque area (F and L) (*P < 0.05, unpaired t test; n = 6). (M) Assay of cytokines by ELISA in mouse brain homogenates revealed reductions in tumor necrosis factor–α (TNF-α), interferon-γ (IFN-γ), and interleukin-12 (IL-12) and an increase in the IL-10/IL-12 ratio in 16-month-old APP/PS1;C3 KO mice compared to APP/PS1 mice (*P < 0.05, independent unpaired t test per marker followed by Bonferroni correction for multiple comparisons; n = 8). KC-GRO, keratinocyte chemoattractant (KC) chemokines CXCL1/2, mouse homologues of human growth-regulated oncogenes (GRO).

  • Fig. 5. C3 deficiency resulted in partial preservation of synapse density in APP/PS1 mice despite an increased plaque load.

    Comparisons were made between WT and C3 KO, WT and APP/PS1, APP/PS1 and APP/PS1;C3 KO, and C3 KO and APP/PS1;C3 KO mice. (A) Synaptic puncta of pre- and postsynaptic markers Vglut2 and GluR1, respectively, and their colocalization in hippocampal CA3 were analyzed by high-resolution confocal microscopy in 16-month-old mice. Scale bar, 5 μm. (B) C3 KO mice had increased Vglut2 and GluR1 synaptic densities compared to WT, APP/PS1, and APP/PS1;C3 KO mice. APP/PS1 mice had fewer GluR1 synaptic densities than WT mice, whereas APP/PS1;C3 KO mice had more GluR1 densities than APP/PS1 mice and were not significantly different from WT mice (*P < 0.05, **P < 0.01, one-way ANOVA and Bonferroni post hoc test per marker; n = 6 to 8; three equidistant planes, 300 μm apart). (C) Colocalization of pre- and postsynaptic puncta revealed increased puncta in C3 KO versus WT and APP/PS1 but not APP/PS1;C3 KO mice, reduced puncta in APP/PS1 mice versus WT mice, and a rescue of synaptic puncta in APP/PS1;C3 KO mice compared to APP/PS1 mice, suggesting partial protection against synapse loss by deletion of C3 (one-way ANOVA and Bonferroni post hoc test). (D and E) Western blotting of synaptic proteins in hippocampal synaptosomes isolated from aged mice indicated increased postsynaptic proteins GluR1, PSD95, and Homer1 in C3 KO versus WT, APP/PS1, and APP/PS1;C3 KO mice. APP/PS1 mice had lower postsynaptic GluR1, PSD95, and Homer1 and presynaptic SYN-1 and SYP compared to WT mice. APP/PS1;C3 KO mice had more GluR1, PSD95, Homer1, SYN-1, and SYP than APP/PS1 mice and were not significantly different than WT mice, suggesting a sparing of synaptic loss in aged C3-deficient APP/PS1 mice (*P < 0.05, **P < 0.01, one-way ANOVA and Bonferroni post hoc test per marker; n = 6). (F and G) Western blotting and quantification of TrkB, mBDNF, CREB, and pCREB in hippocampal homogenates of 16-month-old mice. C3 KO mice had increased mBDNF and pCREB compared to WT, APP/PS1, and APP/PS1;C3 KO mice. APP/PS1 mice showed reductions in all four markers compared to WT mice. APP/PS1;C3 KO mice had higher mBDNF, CREB, and pCREB than APP/PS1 mice (*P < 0.05, **P < 0.01, one-way ANOVA and Bonferroni post hoc test per marker; n = 6), suggesting a partial rescue of age- and AD-related lowering of BDNF pathway proteins. (H) Table summarizing the effects of C3 deficiency on synaptic and BDNF-related proteins. N.S., not significant.

  • Fig. 6. C3 deficiency resulted in partial sparing of neuron loss in hippocampal CA3 in 16-month-old APP/PS1 mice.

    (A) NeuN immunoreactivity in hippocampal CA3, CA1, and dentate gyrus (DG) and prefrontal cortex (PFC) regions in APP/PS1 and APP/PS1;C3 KO mice. Scale bars, 50 μm. (B) APP/PS1;C3 KO mice had more neurons in hippocampal CA3 compared to APP/PS1 mice (*P < 0.05, unpaired t test). (C to E) No significant differences were observed in neuron numbers in CA1, dentate gyrus, and PFC between APP/PS1 and APP/PS1;C3 KO mice. (F) Age-dependent neuron loss was observed between P30 and 16 months of age in WT and APP/PS1 mice and also between 4 and 16 months of age in APP/PS1 mice. However, neuron loss was not observed in C3 KO or APP/PS1;C3 KO mice (*P < 0.05, **P < 0.01, two-way ANOVA and Bonferroni post hoc test; n = 6 to 8; six equidistant planes, 150 μm apart).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/392/eaaf6295/DC1

    Materials and Methods

    Fig. S1. Behavioral testing of locomotion and anxiety.

    Fig. S2. C3 deficiency had no effect on Aβ deposition in young APP/PS1 mice at the earliest stage of plaque deposition.

    Fig. S3. Immunoreactivity of Aβx–42, Aβx–40, and Aβ1–x in aged mice.

    Fig. S4. Glial immunoreactivity was similar in young WT, APP/PS1, and APP/PS1;C3 KO mice at the earliest stage of plaque deposition.

    Fig. S5. C3 deficiency resulted in morphological differences in Iba-1–immunoreactive cells in APP/PS1 aged mice.

    Fig. S6. C3 deficiency resulted in reduced activation of microglia within Aβ plaques in aged APP/PS1 mice but not in aged J20 mice.

    Fig. S7. MAP-2–positive dendrites were better preserved within Aβ plaques in 16-month-old C3-deficient APP/PS1 mice versus APP/PS1 mice.

    Fig. S8. The amounts of APP and PS1 were not significantly different between APP/PS1 and APP/PS1;C3 KO mice.

    References (68, 69)

  • Supplementary Material for:

    Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice

    Qiaoqiao Shi, Saba Chowdhury, Rong Ma, Kevin X. Le, Soyon Hong, Barbara J. Caldarone, Beth Stevens, Cynthia A. Lemere*

    *Corresponding author. Email: clemere{at}bwh.harvard.edu

    Published 31 May 2017, Sci. Transl. Med. 9, eaaf6295 (2017)
    DOI: 10.1126/scitranslmed.aaf6295

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Behavioral testing of locomotion and anxiety.
    • Fig. S2. C3 deficiency had no effect on Aβ deposition in young APP/PS1 mice at the earliest stage of plaque deposition.
    • Fig. S3. Immunoreactivity of Aβx–42, Aβx–40, and Aβ1–x in aged mice.
    • Fig. S4. Glial immunoreactivity was similar in young WT, APP/PS1, and APP/PS1;C3 KO mice at the earliest stage of plaque deposition.
    • Fig. S5. C3 deficiency resulted in morphological differences in Iba-1–immunoreactive cells in APP/PS1 aged mice.
    • Fig. S6. C3 deficiency resulted in reduced activation of microglia within Aβ plaques in aged APP/PS1 mice but not in aged J20 mice.
    • Fig. S7. MAP-2–positive dendrites were better preserved within Aβ plaques in 16-month-old C3-deficient APP/PS1 mice versus APP/PS1 mice.
    • Fig. S8. The amounts of APP and PS1 were not significantly different between APP/PS1 and APP/PS1;C3 KO mice.
    • References (68, 69)

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