A complement–microglial axis drives synapse loss during virus-induced memory impairment

Abstract

Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae1,2. Although thousands of cases of WNV-mediated memory dysfunction accrue annually3, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development4,5. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein6,7 leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34−/− mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease.

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Figure 1: WNV-mediated spatial learning and memory impairments and activated microglia persist beyond 45 days post-infection.
Figure 2: Transcriptional profile of good and poor spatial learners during WNV recovery.
Figure 3: West Nile virus causes a loss in hippocampal CA3 synaptic terminals in mice and humans.
Figure 4: Classical complement activation in neurons and microglia drives WNV-mediated synaptic terminal elimination.

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Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data has been deposited in the Gene Expression Omnibus under the accession number GSE72139.

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Acknowledgements

Funding for this research was provided by the NIH F31 NS077640 (M.J.V.), R01 NS052632 (R.S.K.), and U19 AI083019 (R.S.K. and M.S.S.). The authors would like to thank J. Atkinson and X. Wu for reagents and M. Diamond for critical reading of the manuscript.

Author information

M.J.V. and R.S.K. contributed to the study design. M.J.V., C.G., D.D., D.M.D., B.B., A.S., J.Y., C.P.-T., A.F., D.K.W., K.F., X.J., S.M., J.K.R., J.R.G., R.E.S., B.S. and R.S.K. contributed to data collection and/or interpretation. C.G., J.R.B. and M.S.S. developed single-strand PCR assays for WNV. B.K.D., K.L.T. identified, collected and provided patient samples. M.J.V. and R.S.K. wrote the paper. All authors discussed and commented on the manuscript.

Correspondence to Robyn S. Klein.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Murine intracranial infection with attenuated WNV-NS5-E218A induces similar viral loads and inflammatory response as wild-type WNV-NY99, but greater overall survival.

a, Plaque assay for infectious virus (measured in plaque-forming units per g of tissue) performed on dissected brain tissue at various days post-infection with either footpad infection with 102 pfu of WNV-NY99 or intracranial infection with 104 pfu of WNV-NS5-E218A. Each point represents an individual mouse. b, Survival curves of mice infected at 8-weeks-old by the footpad with WNV-NY99 or intracranially with WNV-NY99 or WNV-NS5-E218A. c, Flow cytometric analysis of dissected cortex, hippocampus and cerebellum at 6 dpi with WNV-NY99 and WNV-NS5-E218A with plots for CD45 and CD11b. d, Quantification of flow cytometry data from c. Shown are numbers of leukocytes (CD45high), lymphocytes (CD45high, CD11blow), and activated macrophages and microglia (CD45high, CD11bhigh) compared to mock-infected controls (n = 4 mice per group). e, Immunostaining and counts for TUNEL staining for apoptotic cells with co-staining for the neuronal marker, NeuN, during peak infection (7 dpi) of WNV-NS5-E218A (n = 5) compared to mock-infected controls (n = 4). DG, dentate gyrus, CTX, entorhinal, perirhinal, and visual cortex. f, Some mice were tested at 22 dpi on a three-day version of the Barnes maze, and evaluated for latency to find target hole (*P < 0.05 by repeated measures two-way ANOVA). g, Prior to Barnes maze testing, mice were tested on open field for total lines crossed in 2 min at 21 dpi. h, qPCR for positive strand (non-replicating strand) and negative strand (replicating) WNV envelope protein message remaining in hippocampal tissue at 7, 25 and 52 dpi (n = 13, 4, and 14 mice per group for 7, 25, and 52 dpi, respectively), measured in copies per Gapdh. i, qPCR for positive strand WNV envelope protein at 52 dpi in WNV good learners (fewer than 8 errors on day 2 of Barnes maze, n = 5) and WNV poor learners (greater than 9.5 errors on day 2 of Barnes maze, n = 9). j, qPCR for negative strand WNV envelope protein at 52 dpi in WNV good learners (fewer than 8 errors on day 2 of Barnes maze, n = 5) and WNV poor learners (greater than 9.5 errors on day 2 of Barnes maze, n = 9). Result was not significant by student’s two-tailed t-test.

Extended Data Figure 2 At 25–52 days post-WNV-NS5-E218A infection, mice do not show any appreciable loss in brain volume, neuron or astrocyte numbers, or macrophage infiltration.

a, Immunostaining for the neuronal marker, NeuN, with TUNEL staining for apoptotic cells within the hippocampus at 52 dpi. Quantification of the number of TUNEL+ neurons and total TUNEL+ cells is shown in mock (n = 3) and WNV-NS5-E218A (n = 6). Scale bar, 20 μm. b, Immunostaining and quantification of the number of NeuN+ neurons per mm2 within the CA1, CA3, dentate gyrus and entorhinal cortex at 25 days after mock (n = 4) or WNV-NS5-E218A infection. WNV-infected animals were subdivided into good (n = 5) and poor (n = 3) learners. Scale bar, 100 μm. c, Post-mortem mouse brains were imaged by MRI at 52 dpi to determine tissue volume of the hippocampus (outlined in red) and total brain (n = 5 mice per group). Scale bar, 1 mm. Not significant by student’s two-tailed t-test (P < 0.05 considered significant). d, Immunostaining for the reactive astrocyte marker, GFAP, shows that WNV-NS5-E218A-infected mice do not exhibit greater hippocampal astrocyte activation than mock-infected controls at 52 dpi. NS, not significant by student’s two-tailed t-test. e, Haematoxylin and eosin (H&E) staining was performed at 52 dpi in WNV-NS5-E218A-recovered and mock-recovered mice. Occasional microglial nodules (arrowhead) surrounded by lymphocytes were observed within the hippocampus. CA1 pyr, CA1 pyramidal layer. f, Flow cytometric analysis of whole brain from mock and WNV-NS5-E218A-infected mice at 8 and 25 dpi was performed to determine numbers of microglia (CD45low, CD11blow), macrophages (CD45high, CD11bhigh), and lymphocytes (CD45high, CD11bnegative). Note the decrease in macrophage population from 7 to 25 dpi.

Extended Data Figure 3 Despite synaptic terminal loss, no changes to synaptic terminal size, axons, or astrocyte or antibody association with terminals during WNV infection.

a, Immunostaining for the presynaptic marker, synaptophysin, at 7 dpi comparing mock (n = 7) with WNV-NS5-E218A-infected (n = 5) mice. Quantification of synaptophysin+ puncta size was performed within the hippocampal CA3. Scale bar, 10 μm. b, Immunostaining for the presynaptic marker, synapsin1, within the hippocampal CA3 in uninfected controls (n = 3) and footpad-infected WNV-NY-1999 (n = 4) at 8 dpi. Quantification was performed on the numbers of synapsin1+ puncta per mm2 with *P < 0.05 considered significant. c, Immunostaining within the hippocampal CA3 for SMI-31, which detect phosphorylated neurofilament and marks axons at 25 dpi (n = 5–6 mice per group). Quantification of the area of SMI-31 per mm2 (not significant by Student’s t-test). d, Immunostaining within the hippocampal CA3 for the presynaptic marker, synaptophysin, co-labelled with the astrocyte marker, S100β at 7 dpi (n = 3 mice per group). Quantification of the percentage of total S100β+ area and synaptophysin+ area colocalized with S100β (not significant by Student’s t-test). e, Electron microscopy was performed on hippocampal CA3 sections from day 7 after mock (left panel) or WNV-NS5-E218A (right panels) infection, with immune-DAB enhancement of IBA1. Note the presence of many phagosomes and cytoplasmic inclusions within the WNV-E218A-infected microglia. Electron micrographs shown are representative of n = 3 mice per group. Scale bars, 1 μm. f, Immunostaining for the presynaptic marker, VGlut1, and endogenous murine IgG (mIgG) at 7 days after mock (n = 4) or WNV-NS5-E218A (n = 4) infection. Quantification was performed on the total per cent of mIgG staining area as well as the per cent of VGlut1+ staining area colocalized with mIgG. g, Immunostaining for the postsynaptic marker, Homer1, and endogenous mIgG at 25 days after mock (n = 4) or WNV-NS5-E218A-infection, which were divided into WNV-infected mice which made fewer than 8 errors on day 2 of the Barnes maze (WNV good learners, n = 5) and WNV-infected mice which made greater than 9.5 errors on day 2 of the Barnes maze testing (WNV poor learners, n = 3). Quantification was performed on the total per cent of mIgG staining area as well as the percent of Homer1+ staining area colocalized with mIgG. Significance was determined by Student’s two-tailed t-test with P < 0.05 considered as significant. NS, not significant. h, Immunostaining and quantification of number of VGlut1 hippocampal CA3 presynaptic terminals at 7 dpi in wild-type and μMT−/− mice. (*P < 0.05, NS, not significant, by Student’s two-tailed t-test). Scale bars, 10 μm.

Extended Data Figure 4 WNV infection of human hippocampal CA2/CA3 neurons with loss of synapses within the hippocampal CA1 and the entorhinal cortex.

a, Immunostaining of human WNV encephalitis and control post-mortem hippocampal tissue for WNV-antigen. Shown at high magnification are neuron cell bodies (arrows) and neurites (arrowheads) within the hippocampal CA2/CA3 region. b, c, Immunostaining within the hippocampal CA1 (b) or entorhinal cortex (c) for the presynaptic marker, synaptophysin, within human WNV encephalitis and control autopsy cases. Quantification of the per cent of synaptophysin+ area (hippocampal CA1 P = 0.3, entorhinal cortex P = 0.11 by two-tailed Student’s t-test (not significant). Scale bar, 20 μm. In one WNV encephalitis patient sample, the entorhinal cortex could not be quantified because it was missing from the section.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-6. (PDF 253 kb)

41586_2016_BFnature18283_MOESM85_ESM.mov

Video of Barnes Maze day 2, trial 2 in mock‐infected mouse at day 47 post‐infection. Number of errors before finding target hole and latency to find target hole was quantified in Figure 1. (MOV 1905 kb)

Video 1: Barnes Maze day 2, trial 2 in mock‐infected mouse at day 47 post‐infection

Video of Barnes Maze day 2, trial 2 in mock‐infected mouse at day 47 post‐infection. Number of errors before finding target hole and latency to find target hole was quantified in Figure 1. (MOV 1905 kb)

41586_2016_BFnature18283_MOESM86_ESM.mov

Video of Barnes Maze day 2, trial 2 in WNV‐NS5‐E218A‐recovered mouse at day 47 post‐infection. Number of errors before finding target hole and latency to find target hole was quantified in Figure 1. (MOV 10337 kb)

Video 2: Barnes Maze day 2, trial 2 in WNV‐NS5‐E218A‐recovered mouse at day 47 post‐infection

Video of Barnes Maze day 2, trial 2 in WNV‐NS5‐E218A‐recovered mouse at day 47 post‐infection. Number of errors before finding target hole and latency to find target hole was quantified in Figure 1. (MOV 10337 kb)

41586_2016_BFnature18283_MOESM87_ESM.mov

This video shows a 3D reconstruction from confocal Z‐stack images of immunostaining of CX3CR1‐GFP and the presynaptic marker, synaptophysin, in CX3CR1‐GFP +/‐ mice in hippocampus of mock‐infected control mouse at day 7 post‐infection. (MOV 1692 kb)

Video 3: 3D reconstruction from confocal Z‐stack images of immunostaining of CX3CR1‐GFP and the presynaptic marker, synaptophysin, in CX3CR1‐GFP +/‐ mice in hippocampus of mock‐infected control mouse at day 7 post‐infection.

This video shows a 3D reconstruction from confocal Z‐stack images of immunostaining of CX3CR1‐GFP and the presynaptic marker, synaptophysin, in CX3CR1‐GFP +/‐ mice in hippocampus of mock‐infected control mouse at day 7 post‐infection. (MOV 1692 kb)

41586_2016_BFnature18283_MOESM88_ESM.mov

This video shows a 3D reconstruction from confocal Z‐stack images of immunostaining of CX3CR1‐GFP and the presynaptic marker, synaptophysin, in CX3CR1‐GFP +/‐ mice in hippocampus of WNV‐NS5‐E218A‐infected mouse at day 7 post‐infection. (MOV 2433 kb)

Video 4: 3D reconstruction from confocal Z‐stack images of immunostaining of CX3CR1‐GFP and the presynaptic marker, synaptophysin, in CX3CR1‐GFP +/‐ mice in hippocampus of WNV‐NS5‐E218A‐infected mouse at day 7 post‐infection.

This video shows a 3D reconstruction from confocal Z‐stack images of immunostaining of CX3CR1‐GFP and the presynaptic marker, synaptophysin, in CX3CR1‐GFP +/‐ mice in hippocampus of WNV‐NS5‐E218A‐infected mouse at day 7 post‐infection. (MOV 2433 kb)

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Vasek, M., Garber, C., Dorsey, D. et al. A complement–microglial axis drives synapse loss during virus-induced memory impairment. Nature 534, 538–543 (2016). https://doi.org/10.1038/nature18283

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