Question Journal Article Perspective Papers Do the summary statement of the paper attached in not more than one page For your summary statement, address the following questions: - What specific question/hypothesis were the researchers attempting to address? - What are the most important experiments in the paper that addressed this question? (DO NOT try to discuss all the experiments—choose the ones that you feel are most important) - What methods/techniques did the researchers use in these experiments? - What conclusions did the authors draw based on these results? - How did the conclusions address the researchers’ original question/hypothesis? Instructions: Answer all the questions following the instructions. Strictly do not use AI for solving questions. The solution should be free of Plagiarism. Solution to be formatted in APA and use appropriate references with in-text citations in APA./nRESEARCH | REPORTS 2. B. Bosworth, K. Zhang, "Evidence of Increasing Differential Mortality: A Comparison of the HRS and SIPP," Center for Retirement Research at Boston College Working Paper 2015-13 (2015). 3. R. Chetty et al., JAMA 10.1001/jama.2016.4226 (2016). 4. National Research Council, Committee on the Long-Run Macroeconomic Effects of the Aging U.S. Population, "The Growing Gap in Life Expectancy by Income: Implications for Federal Programs and Policy Responses" (2015). 5. J. Pijoan-Mas, J. V. Ríos-Rull, Demography 51, 2075-2102 (2014). 6. H. Waldron, Soc. Secur. Bull. 67, 1-28 (2007). 7. H. Waldron, Soc. Secur. Bull. 73, 1-37 (2013). 8. J. Wilmoth, C. Boe, M. Barbieri, in International Differences in Mortality at Older Ages: Dimensions and Sources, E. M. Crimmins, S. H. Preston, B. Cohen, Eds. (National Academies Press, Washington, DC, 2011), pp. 337-372. 9. G. K. Singh, M. Siahpush, Int. J. Epidemiol. 35, 969-979 (2006). 10. M. Ezzati, A. B. Friedman, S. C. Kulkarni, C. J. Murray, PLOS Med. 5, e66 (2008). 11. C. J. Murray et al., PLOS Med. 3, e260 (2006). 12. H. Wang, A. E. Schumacher, C. E. Levitz, A. H. Mokdad, C. J. Murray, Popul. Health Metr. 11, 8 (2013). 13. J. S. Olshansky et al., Health Aff. 31, 1803-1813 (2011). 14. E. R. Meara, S. Richards, D. M. Cutler, Health Aff. 27, 350-360 (2008). 15. D. M. Cutler, F. Lange, E. Meara, S. Richards-Shubik, C. J. Ruhm, J. Health Econ. 30, 1174-1187 (2011). 16. J. K. Montez, L. F. Berkman, Am. J. Public Health 104, e82-e90 (2014). 17. Human Mortality Database; www.mortality.org. 18. D. D. Reidpath, P. Allotey, J. Epidemiol. Community Health 57, 344-346 (2003). 19. A. Case, A. Deaton, Proc. Natl. Acad. Sci. U.S.A. 112, 15078-15083 (2015). 20. J. Bound, A. Geronimus, J. Rodriguez, T. Waidman, "The Implications of Differential Trends in Mortality for Social Security Policy," University of Michigan Retirement Research Center Working Paper 2014-314 (2014). 21. J. B. Dowd, A. Hamoudi, Int. J. Epidemiol. 43, 983-988 (2014). 22. T. Goldring, F. Lange, S. Richards-Shubik, "Testing for Changes in the SES-Mortality Gradient When the Distribution of Education Changes Too," National Bureau of Economic Research Working Paper 20993 (2015). 23. A. S. Hendi, Int. J. Epidemiol. 44, 946-955 (2015). 24. A. Aizer, J. Currie, Science 344, 856-861 (2014). 25. D. Brown, A. Kowalski, I. Lurie, "Medicaid as an Investment in Children: What Is the Long-Term Impact on Tax Receipts?" National Bureau of Economic Research Working Paper 20835 (2015). 26. S. Cahodes, S. Kleiner, M. F. Lovenhem, M. Grossman, "Effect of Child Health Insurance Access on Schooling." National Bureau of Economic Research Working Paper 20178 (2014). 27. S. Miller, L. R. Wherry, "The Long-Term Health Effects of Early Life Medicaid Coverage," Social Science Research Network Working Paper 2466691 (2015). 28. L. R. Wherry, B. Meyer, "Saving Teens: Using and Eligibility Discontinuity to Estimate the Effects of Medicaid Eligibility." National Bureau of Economic Research Working Paper 18309 (2013). 29. L. R. Wherry, S. Miller, R. Kaestner, B. D. Meyer, "Childhood Medicaid Coverage and Later Life Health Care Utilization," National Bureau of Economic Research Working Paper 20929 (2015). 30. J. Ludwig, D. L. Miller, Q. J. Econ. 122, 159-208 (2007). 31. H. Hoynes, D. Whitmore-Schanzanbach, D. Almond, "Long Run Impacts of Childhood Access to the Safety Net," National Bureau of Economic Research Working Paper 18535 (2012). 32. A. Isen, M. Rossin-Slater, R. Walker, "Every Breath You Take Every Dollar You'll Make: The Long-Term Consequences of the Clean Air Act of 1970," National Bureau of Economic Research Working Paper 19858 (2014). 33. A. Fenelon, S. H. Preston, Demography 49, 797-818 (2012). 34. D. de Walque, J. Hum. Resour. 45, 682-717 (2010). 35. C. E. Finch, E. M. Crimmins, Science 305, 1736-1739 (2004). ACKNOWLEDGMENTS We thank M. Barbieri, A. Case, A. Deaton, J. Goldstein, I. Kuziemko, R. Lee, and K. Wachter, as well as seminar participants at Berkeley, the Chicago Federal Reserve, Fundação Getúlio Vargas São Paulo, Bonn University, University of Munich, Princeton University, ETH Zurich, and the University of Zurich for comments. Supported by Princeton Center for Translational Research on Aging grant 2P30AG024928. Data and code are available at http://dx.doi.org/10.7910/DVN/C2VYNM. SUPPLEMENTARY MATERIALS www.sciencemag.org/content/352/6286/708/suppl/DC1 Materials and Methods Figs. S1 to S8 Tables S1 to S4 References (36-38) 22 December 2015; accepted 17 March 2016 Published online 21 April 2016 10.1126/science.aaf1437 NEURODEVELOPMENT Complement and microglia mediate early synapse loss in Alzheimer mouse models Soyon Hong,1 Victoria F. Beja-Glasser,1* Bianca M. Nfonoyim,1* Arnaud Frouin,1 Shaomin Li,2 Saranya Ramakrishnan,1 Katherine M. Merry,1 Qiaogiao Shi,2 Arnon Rosenthal, 3,4,5 Ben A. Barres,6 Cynthia A. Lemere,2 Dennis J. Selkoe,2,7 Beth Stevens1,8+ Synapse loss in Alzheimer's disease (AD) correlates with cognitive decline. Involvement of microglia and complement in AD has been attributed to neuroinflammation, prominent late in disease. Here we show in mouse models that complement and microglia mediate synaptic loss early in AD. C1q, the initiating protein of the classical complement cascade, is increased and associated with synapses before overt plaque deposition. Inhibition of C1q, C3, or the microglial complement receptor CR3 reduces the number of phagocytic microglia, as well as the extent of early synapse loss. C1q is necessary for the toxic effects of soluble ß-amyloid (AB) oligomers on synapses and hippocampal long-term potentiation. Finally, microglia in adult brains engulf synaptic material in a CR3-dependent process when exposed to soluble Aß oligomers. Together, these findings suggest that the complement-dependent pathway and microglia that prune excess synapses in development are inappropriately activated and mediate synapse loss in AD. Downloaded from http://science.sciencemag.org/ on January 2, 2020 G enome-wide association studies impli- cate microglia and complement-related pathways in Alzheimer's disease (AD) (1). Previous research has demonstrated both beneficial and detrimental roles of com- plement and microglia in plaque-related neuro- pathology (2, 3); however, their roles in synapse loss, a major pathological correlate of cognitive decline in AD (4), remain to be identified. Emerg- ing research implicates microglia and immune- related mechanisms in brain wiring in the healthy 1F.M. Kirby Neurobiology Center, Boston Children's Hospital (BCH) and Harvard Medical School (HMS), Boston, MA 02115, USA. 2Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital (BWH) and HMS, Boston, MA 02115, USA. 3Alector Inc., 953 Indiana Street, San Francisco, CA 94107, USA. 4Annexon Biosciences, 280 Utah Avenue Suite 110, South San Francisco, CA 94080, USA. 5Department of Anatomy, University of California San Francisco (UCSF), San Francisco, CA 94143, USA. Department of Neurobiology, Stanford University School of Medicine, Palo Alto, CA 94305, USA. 7Prothena Biosciences, Dublin, Ireland. "Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. *These authors contributed equally to this work. +Corresponding author. Email: beth.stevens@childrens.harvard.edu brain (1). During development, C1q and C3 local- ize to synapses and mediate synapse elimination by phagocytic microglia (5-7). We hypothesized that this normal developmental synaptic pruning pathway is activated early in the AD brain and mediates synapse loss. The degree of region-specific synapse loss is a stronger correlate of cognitive decline in AD than counts of plaques, tangles, and neuronal loss (8, 9). To determine how early synapse loss occurs, we used superresolution structured illu- mination microscopy (SIM) (10) to quantify syn- apse density in hippocampal CA1 stratum radiatum of familial AD-mutant human amyloid precursor protein (hAPP) ("J20") transgenic mice (11). Quan- tification of colocalized pre- and postsynaptic puncta [synaptophysin and postsynaptic den- sity 95 (PSD95) (Fig. 1A); synaptotagmin and homer (fig. S1, A to D)] revealed a significant loss of synapses in J20 hippocampus at 3 to 4 months old (mo), an age that precedes plaque deposition (11, 12). Synapse loss in preplaque J20 CA1 was confirmed by electron microscopy (fig. S1G). Con- focal imaging also showed synapse loss in CA1, CA3, and dentate gyrus of 3 mo J20 hippo- campus but not in striatum (fig. S1E). Synapse 712 6 MAY 2016 . VOL 352 ISSUE 6286 sciencemag.org SCIENCE RESEARCH | REPORTS A WT J20 Synaptophysin + PSD95 Physin PSD95 150 Individual or Colocalized Puncta *** *** WT 100- % WT 50- 0 J20 B DG FC STR CRB Physin PSD95 Physin+PSD95 C1q DAP 150- *** WT WT J20 *** 100- 50- ns J20 C1q Intensity Levels (A.U.) T ns T 0 DG FC STR CRB C C1q PSD95 D C1g PSD95 % PSD95 Colocalized with C1q 250- * 200- Downloaded from http://science.sciencemag.org/ on January 2, 2020 % WT 150- 100- 50 0 WT J20 E WT J20 WT J20 Aß DAPI C1g Aß Levels in J20 DG C1q Levels in J20 DG 150- 150- * Veh % Vehicle-Rx ** 100- T % Vehicle-Rx $ 100- 50 50- CpdE 0 0 Veh CpdE Veh CpdE Fig. 1. C1q up-regulation and deposition onto synapses precede pre- plaque synapse loss in J20 mice. (A) Superresolution SIM images of synaptophysin (green)- and PSD95 (red)-immunoreactive puncta in stratum radiatum of 3 mo J20 or WT hippocampus (CA1). Quantification of synaptic puncta or their apposition using Imaris indicates selective loss of PSD95 in J20 hippocampus as compared to their WT littermate controls. See fig. S1. (B) Region-specific up-regulation of C1q (green) in 1 mo J20; DG, dentate gyrus; FC, frontal cortex; STR, striatum; CRB, cerebellum; DAPI, 4',6-diamidino-2-phenylindole. See fig. S2. (C) Orthogonal view of SIM image showing colocalization of C1q (green) and PSD95 (red). (D) Higher percentage of PSD95 colocalized with C1q in 1 mo J20 dentate gyrus versus WT. (E) Compound E reduces deposited soluble Aß (red) and Clq (green) in 1 mo J20 dentate gyrus, with minimal effect on C1q levels in WTmice. Scale bar, 2 um (A, C, and D) or 10 um (B and E). Means ± SEM; n = 3 or 4 mice per genotype or per treatment group per genotype. * P < 0.05, ** P < 0.01, or *** P < 0.001 using two-way analysis of variance (ANOVA) followed by Bonferroni posttest (A and B), two-tailed one-sample t test (D), or two- tailed unpaired t test (E). levels were not altered in 1 mo J20 brains ver- sus wild-type (WT) littermates (fig. S1F), sug- gesting that the hippocampal synaptic loss at 3 mo is likely not a result of abnormal synaptic development. We asked whether the classical complement cascade is up-regulated in preplaque brains when synapses are already vulnerable. C1q immuno- reactivity (13) (antibody now available at Abcam) was elevated in J20 brains as early as 1 mo and preceding synapse loss (Fig. 1B and fig. S1). C1q elevation was region-specific, particularly in the hippocampus and frontal cortex, two regions vulnerable to synapse loss (14) (Fig. 1B and fig. S2A). C1q immunoreactivity was comparable be- tween J20 and WT mice at postnatal day 21 (P21) (fig. S2B), suggesting that elevated levels at 1 mo are likely not a developmental artifact. C1q was also similarly increased in the hippocampus of another model of AD, the APP/PS1 (presenilin 1) mice (15) (fig. S2C). Notably, SIM demonstrated colocalization of C1q with PSD95-positive puncta in 1 mo J20 hippocampus (Fig. 1C). A higher percentage of PSD95 colocalized with C1q in the hippocampus of J20 mice than in that of WT littermates (Fig. 1D and fig. S3), suggesting that the C1q-associated synapses may be marked for elimination. Punctate Aß was found deposited in J20 hip- pocampus at 1 mo (fig. S4), long before Aß plaques deposit (11, 12), raising the question of whether C1q increase in these preplaque brains is dependent on soluble Aß levels. To test this hypothesis, we injected the mice with compound E, a y-secretase inhibitor that rapidly decreases Aß production (12). Compound E markedly re- duced soluble Aß levels in J20 mice; there was a corresponding reduction of C1q deposition (Fig. 1E), suggesting that Aß up-regulates Clq. SCIENCE sciencemag.org 6 MAY 2016 . VOL 352 ISSUE 6286 713 RESEARCH | REPORTS A Control Aß Monomer Aß Oligomer B C1q PSD95 % PSD95 Colocalized With C1q C1q 250- * 200- % WT 150- 100- 50- 0 Mon. Rx Olig. Rx C Control Aß Monomer Aß Oligomer % CD68 Occupancy in C1qa WT Microglia CD68 60- ** Iba1 % Total Microglia ## Control Rx AB Monomer Rx 40- AB Oligomer Rx T 20- 0 0 1 2 3 100- I D % Total Microglia % CD68 Occupancy in C1qa KO Microglia Control Rx 80- JAB Monomer Rx 60- Aß Oligomer Rx Downloaded from http://science.sciencemag.org/ on January 2, 2020 Fig. 3. Complement is necessary for synapse loss and dysfunction in AD models. (A) Aß oli- gomers induced loss of colocalized synapsin- and PSD95-immunoreactive puncta in the contralateral hippocampus of 3 mo WT mice (left panel); however, they failed to do so in C1qa KO mice (right panel). (B) Coinjection of Aß oligomers with the function- blocking antibody against C1q, ANX-M1, but not with its IgG isotype control, prevented synapse loss in WT mice. (C) Pretreatment of hippocampal slices with the anti-Clq antibody, ANX-M1, prevented Aß- mediated LTP inhibition (green) versus IgG (red). IgG alone had a minimal effect (blue) versus artificial cerebrospinal fluid (aCSF) vehicle (black). n = 6 to 11 slices per group. (D) Percentage of PSD95 co- localized with C3 is increased in APP/PS1 hippo- campus versus that of WT mice. (E and F) Genetic deletion of C3 prevents synapse loss in 4 mo APP/ PS1 mice. Quantification of colocalized immuno- reactive puncta for synaptotagmin and homer in dentate gyrus (E) or synaptophysin and PSD95 in CA1 stratum radiatum (F) of WT, APP/PS1, APP/ PS1xC3 KO, and C3 KO hippocampi. Means ± SEM; n = 3 to 5 mice per genotype or per treatment group per genotype. * P < 0.05, ** P < 0.01, or *** P < 0.001 using two-tailed one-sample t test (D), one- way (A, C, E, F) or two-way (B) ANOVA followed by Bonferroni posttest. ns, not significant. A Synapsin + PSD95 Colocalized Puncta 40- 20- 2 NOCH 1 0 0 1 T 3 %CD68 within Iba1+ Microglia Fig. 2. Oligomeric AB increases Clq and microglial phagocytic activity. (A and B) Soluble AB oligomers in WT mice led to elevation of Clq (green) (A) and a higher percentage of PSD95 (red) colocalization with Clq versus monomers (B). (C and D) oAß induced high levels of CD68 (green) immunoreactivity in Ibal-positive (red) microglia in WTmice (C), but not in those of C1qa KO mice (D). Both had negligible changes in morphology. See fig. S10. Scale bar, 10 um (A), 5 um (B), or 20 um (C). Means ± SEM; n = 3 to 5 mice per treatment group per genotype. * P < 0.05 using two-tailed t test (B) or *P < 0.05, ** P < 0.01 versus control-treated or ##P < 0.01 versus Aß monomer-treated using two-way ANOVA followed by Bonferroni posttest (C). B Synapsin + PSD95 Colocalized Puncta in WT Mice 400- 200- 200- ns Control ns lgG Ctrl % Control 150- 150- Aß Mon. % Control Aß Olig. % oAB-Rx 300- C1q Ab * ** 100- 100- T 200- 50- 50- 100- 0 0 0 C1qa WT C1qa KO PBS oAB PBS OAB C 250- aCSF +- IgG+aCSF % Baseline at 55 min of LTP Induction -IgG+oAB fEPSP slope (%) 200 C1qAb+oAB 200- LTP Magnitudes 180- ns *** 150 *** 160- 140- 100- 120- HFS 50- 100- -20 0 20 40 60 aCSF lgG+ lgG C1q Ab Time (min) aCSF +0Aß +0Aß D % PSD95 Coloc. With C3 Synaptotagmin + Homer in DG F Synaptophysin + PSD95 in CA1 250- * 150 ns 150 ns ns 200- ns * * % WT 150- % WT 100- % WT 100- 100- 50- 50- 50- O 0 WT APP/PS1 WT APP/PS1 APP/PS1 C3 KO WT APP/PS1APP/PS1 C3 KO 0 xC3 KO xC3 KO 714 6 MAY 2016 . VOL 352 ISSUE 6286 sciencemag.org SCIENCE RESEARCH | REPORTS A B Homer-GFP Homer-GFP Iba1 Iba1 15- % Engulfment *** 10- 5. 0- Aß Mon. AB Olig. C % Homer-GFP Engulfment by Microglia D Synaptotagmin + Homer Colocalized Puncta 250- 250- PBS 150- 150- PBS * 200- 200- oAB ns OAß % PBS-Rx % PBS-Rx % PBS-Rx ** % PBS-Rx 150- 150- 100- 100- ns 100- 100- 50- 50- 50- 50- 0 0 Homer-GFP Homer-GFP r0 0- xCR3 WT xCR3 KO To further address whether the increase of C1q is dependent on soluble Aß, and if so, which species, we injected soluble Aß oligomers or monomers into lateral ventricles of WT mice. Hippocampus contralateral to the injection site was examined to avoid any surgery-related ef- fects. Oligomeric Aß (oAß), which is prefibrillar in nature and acts as a mediator of synapse loss and dysfunction in AD (4), but not the relatively innocuous monomeric Aß or vehicle, induced C1q deposition (Fig. 2A and fig. S5). A higher percentage of PSD95 colocalized with C1q in oAß-injected versus monomer-injected mice (Fig. 2B), in a manner similar to this colocalization in J20 mice. Together, these findings show an early and aberrant increase and synaptic localization of C1q in multiple AD model systems. Further- more, fluorescent in situ hybridization (FISH) demonstrated up-regulated C1qa expression in microglia (fig. S6), implicating microglia as a major source of C1q in these preplaque brains. To test whether Clq and oAß act in a common pathway to eliminate synapses, we injected oAß into lateral ventricles of C1qa knockout (KO) mice (16). Soluble oAß induced a significant loss of co- localized synapsin- and PSD95-immunoreactive puncta in WT mice within 72 hours (Fig. 3A, left panel) (17). In contrast, oAß failed to induce syn- apse loss in C1qa KO mice (Fig. 3A, right panel), suggesting that Clq is required for oAß-induced synapse loss in vivo. To determine whether local, acute inhibition of C1 activation could similarly blunt the synaptotoxic effects of oAß, we used an antibody against C1q (anti-C1q) (ANX-M1, Annexon Biosciences), which blocks the classical complement cascade (see fig. S7 and supplemen- tary methods). Coadministration of the ANX-M1 anti-C1q antibody, but not its immunoglobulin G (IgG) isotype control, prevented oAß from inducing synapse loss in WT mice (Fig. 3B). Thus, block- ing C1 activation by either genetic or antibody- mediated means lessened oAB's synaptotoxic effects. To determine whether C1q is associated with synaptic dysfunction, we asked whether the established ability of oAß to potently inhibit long-term potentiation (LTP) (4) was depen- dent on C1q. We tested the functional effects of the ANX-M1 anti-C1q antibody in acute hippo- campal slices treated with oAB. IgG alone had negligible effects on LTP induction in WT mouse hippocampal slices and on the ability of oAß to inhibit LTP; however, pretreatment of hippo- campal slices with the anti-C1q antibody signif- icantly prevented the impairment of LTP by oAß (Fig. 3C). Neither ANX-M1 nor its IgG control altered basal synaptic neurotransmission (fig. S8). Collectively, these results in hippocampal slices and in mice support C1q as a key mediator of oAß-induced synaptic loss and dysfunction. In the healthy developing brain, C1q promotes activation of C3, which opsonizes subsets of synapses for elimination, a process that is down- regulated in the mature brain (5, 6). However, oAß induced a significant C3 deposition in WT adult mice (fig. S7A, upper panel). This was sig- nificantly reduced in both the C1qa KO (fig. S7A, lower panel) and the ANX-M1 anti-Clq antibody- treated WT mice (fig. S7B), suggesting that the C3 deposition in this model is downstream of the classical complement cascade. Consistent with these findings, a higher percentage of PSD95 colocalized with C3 in J20 and APP/PS1 brains (Fig. 3D and fig. S9). To determine whether C3 is necessary for early synapse loss in AD genetic models, we crossed APP/PS1 mice, which, simi- lar to the J20 mice, had a significant increase and localization of C1q and C3 onto hippocampal synapses (figs. S2C and S9), to C3-deficient mice (18). Quantification of colocalized pre- and post- synaptic puncta demonstrated synapse loss in 4 mo APP/PS1 hippocampus as compared to WT; however, APP/PS1xC3 KO mice did not display this synapse loss (Fig. 3, E and F). Together, our data indicate that genetic deletion of C3 amelio- rates synapse loss in APP/PS1 mice, providing further evidence that the classical complement cascade mediates early synapse loss in AD mouse models. Downloaded from http://science.sciencemag.org/ on January 2, 2020 CR3 WT CR3 KO Fig. 4. Microglia engulf synapses via CR3 upon oligomeric Aß challenge. (A) Orthogonal view of high-resolution confocal image shows colocalization of homer-GFP and Iba1 (red). (B) Three-dimensional reconstruction and surface rendering using Imaris demonstrate larger volumes of homer-GFP puncta inside microglia of oAß-injected contralateral hippo- campus versus those of monomer-injected. (C) Mi- croglia of homer-GFPxCR3 KO mice (right panel) show less engulfment of homer-GFP when chal- lenged with oAB versus those of homer-GFP mice (left panel). (D) Aß oligomers failed to induce syn- apse loss in the contralateral hippocampus of CR3 KO mice (right panel) as they did in WT mice (left panel). Scale bar, 5 um (A and B). Means ± SEM; n = 3 mice per treatment group per genotype (n = 6 to 17 microglia analyzed per mouse). * P < 0.05, ** P < 0.01, or *** P < 0.0001 using two-tailed t test (B) or two-tailed one-sample t test (C and D). ns, not significant. Microglia express complement receptors and mediate synaptic pruning in the developing brain (1, 6), raising the question of whether this normal developmental pruning pathway could be acti- vated to mediate synapse loss in the preplaque AD brain. Consistent with this hypothesis, mi- croglia had increased amounts of the lysosomal protein CD68 in J20 hippocampus compared to WT and less so in striatum, a less vulnerable region (figs. S1C and S10). Furthermore, in WT mice challenged with oAß, microglia had sig- nificantly increased levels of CD68 immuno- reactivity (Fig. 2C). However, in C1qa KO mice in which synapse loss was rescued, oAß failed to induce such an increase (Fig. 2D), suggesting that microglia eliminate synapses through the complement pathway. To directly test whether phagocytic microg- lia engulf synaptic elements, we adapted our in vivo synaptic engulfment assay (19) using in- tracerebroventricular injections of Aß in homer- GFP (green fluorescent protein) mice (20) (Fig. 4.A). oAß induced a significantly higher volume of internalized homer-GFP in microglia than monomeric Aß controls did at the contralateral hippocampus (Fig. 4B), indicating that microglia engulf synaptic elements when challenged with oAB. Internalized homer-GFP often colocalized SCIENCE sciencemag.org 6 MAY 2016 . VOL 352 ISSUE 6286 715 RESEARCH | REPORTS with CD68 (fig. S11A), suggesting that the en- gulfed synapses are internalized into lysosom- al compartments in a manner similar to that of developmental synaptic pruning (6). Notab- ly, oAß failed to increase synaptic engulfment in microglia lacking CR3 (21), a high-affinity receptor for C3 expressed on macrophages [homer-GFPxCR3 KO versus homer-GFP mice, which received tail vein injections of phosphate- buffered saline (PBS) or oAß (Fig. 4C)]. These data demonstrate that CR3 is necessary for oAß-dependent engulfment of synapses by microglia. To test whether inhibition in microglial en- gulfment leads to protection against oAß-induced synapse loss, we performed tail vein injections of oAB into WT and CR3 KO mice. oAß induced synapse loss in the hippocampus of WT mice but not in that of CR3 KO mice (Fig. 4D). All CR3-positive microglia were P2RY12-positive (fig. S11), indicating that they are resident cells (22). Altogether, these results suggest that resi- dent microglia engulf synaptic material when chal- lenged by oAß through a complement-dependent mechanism. Synaptic deficits occur in early AD and mild cognitive impairment before onset of plaques and are some of the first signs of the neuronal de- generative process (4, 23-25). Here we identify critical synaptotoxic roles of complement and microglia in AD models before plaque forma- tion and neuroinflammation, in regions of the hippocampus undergoing synapse loss. Using multiple experimental approaches, we demon- strate a region-specific increase of phagocytic microglia and accumulation of C1q and C3 on synapses in preplaque brains. Microglia in the adult brain, when challenged with synapto- toxic, soluble Aß oligomers, engulf synapses in the absence of plaque aggregates; deletion of CR3 blocks this process. Finally, inhibiting C1q, C3, or CR3 activity rescues synaptic loss and dysfunction. Our data suggest a local activation of a de- velopmental pruning pathway (5, 6) as a key mechanism underlying oAß-induced synapse loss in preplaque AD brain. C1q is aberrantly increased by diffusible oAß in a region-specific manner and deposits onto synapses, triggering the activation of downstream classical comple- ment pathway and phagocytic microglia. Block- ing Aß production in J20 mice significantly ameliorated C1q deposition in the hippocampus, and genetic or antibody-mediated inhibition of complement blocks oAß from inducing microg- lial synaptic engulfment, synapse loss, and LTP inhibition. These complementary findings have direct therapeutic relevance. We propose a model in which Clq and oAß operate in a common pathway to activate the complement cascade and drive synapse elimi- nation by microglia through CR3 (fig. S12). This could occur in multiple ways: Soluble oAß asso- ciates with synaptic membranes and other syn- aptic markers (4, 26); thus, oAß bound to synapses may anchor Clq directly. Alternatively, oAß binding to synapses may weaken the synapse (4) and expose a C1q receptor. Although spe- cific receptors for C1q at synapses are not yet known, we have shown that C1q binds syn- apses in vulnerable regions undergoing syn- apse loss (5, 27). It is also plausible that oAß and C1q may work indirectly to mediate syn- apse loss through cytokines such as trans- forming growth factor-ß (7), through microglial or astrocytic activation, or through other mech- anisms, including major histocompatibility complex class I (MHCI)-PirB, another immune pathway critical for synapse elimination in de- velopment and AD (28-30). Finally, our studies show that resident mi- croglia in the adult central nervous system phagocytose synapses when challenged by syn- aptotoxic oAß, implicating microglia as poten- tial cellular mediators of synapse loss. Although microglia and complement activation are pro- minently involved in plaque maintenance and related periplaque neuropathology, their roles have heretofore been largely regarded as a sec- ondary event related to neuroinflammation (2). Our studies directly challenge this view and sug- gest that microglia and immune-related path- ways can act as early mediators of synapse loss and dysfunction that occur in AD models be- fore plaques form. Although the complement pathway may not be involved in all patholog- ical routes to AD, including plaque-associated synapse loss, the work reported here provides new insights into how synapses are lost in AD. It will be important in future studies to examine whether this microglia or the complement- dependent pathway also plays a role in plaque- associated synapse loss or in other synaptopathies, including tauopathies and Huntington's dis- ease. If so, our findings may suggest comple- ment and microglia as potential early therapeutic targets in AD and other neurodegenerative dis- eases involving synaptic dysfunction and memory decline. REFERENCES AND NOTES 1. S. Hong, L. Dissing-Olesen, B. Stevens, Curr. Opin. Neurobiol. 36, 128-134 (2016). 2. T. Wyss-Coray, J. Rogers, Cold Spring Harb. Perspect. Med. 2, a006346 (2012). 3. M. E. Benoit et al., J. Biol. Chem. 288, 654-665 (2013). 4. L. Mucke, D. J. Selkoe, Cold Spring Harb. Perspect. Med. 2, a006338 (2012). 5. B. Stevens et al., Cell 131, 1164-1178 (2007). 6. D. P. Schafer et al., Neuron 74, 691-705 (2012). 7. A. R. Bialas, B. Stevens, Nat. Neurosci. 16, 1773-1782 (2013). 8. S. T. DeKosky, S. W. Scheff, Ann. Neurol. 27, 457-464 (1990). 9. R. D. Terry et al., Ann. Neurol. 30, 572-580 (1991). 10. S. Hong, D. Wilton, B. Stevens, D. S. Richardson, Structured Illumination Microscopy for the investigation of synaptic structure and function. 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Aging 27, 1372-1384 (2006). 25. S. W. Scheff, D. A. Price, F. A. Schmitt, S. T. DeKosky, E. J. Mufson, Neurology 68, 1501-1508 (2007). 26. S. Hong et al., Neuron 82, 308-319 (2014). 27. A. H. Stephan, B. A. Barres, B. Stevens, Annu. Rev. Neurosci. 35, 369-389 (2012). 28. A. Datwani et al., Neuron 64, 463-470 (2009). 29. T. Kim et al., Science 341, 1399-1404 (2013). 30. H. Lee et al., Nature 509, 195-200 (2014). ACKNOWLEDGMENTS We thank B. Sabatini (HMS), T. Bartels (BWH), and members of the Stevens laboratory for critical reading of the manuscript; L. Dissing-Olesen (BCH) for help with the conceptual figure (fig. S12), M. Ericsson [HMS electron microscopy (EM) facility] for EM imaging, K. Kapur (BCH) for advice on statistics, D. M. Walsh (BWH) for Aß oligomers (S26C), S. Okabe (University of Tokyo) for homer-GFP mice, and M. Leviten and T. Yednock (Annexon Biosciences) for characterization and advice on the ANX-M1 anti-C1q antibody; D. Richardson (Harvard Center for Biological Imaging), A. Hill BCH Intellectual and Developmental Disabilities Research Center Cellular Imaging Core NIH-P30-HD-18655, and H. Elliot and T. Xie (HMS Image and Data Analysis Core) for assistance with imaging and data analysis; and S. Kim (BWH), K. Colodner (BCH), and S. Matousek (BWH) for assistance with mice. The J20 mice, C1qa KO mice, P2RY12 antibody, and the ANX-M1 C1q function-blocking antibody are available from L. Mucke, M. Botto, O. Butovsky, and A. Rosenthal under material transfer agreements with UCSF Gladstone, Imperial College London, BWH, and Annexon Biosciences, respectively. A.R. is a cofounder, consultant, and chairman of the board of directors; B.A.B. is a cofounder and chairman of the scientific advisory board; and B.S. serves on the scientific advisory board of Annexon LLC. A.R., B.A.B., and B.S. are minor shareholders of Annexon LLC. All other authors declare no competing financial interests related to this project. The following patents related to this project have been granted or applied for: PCT/2015/010288 (S.H. and B.S.), US14/988387 and EP14822330 (S.H., A.R., and B.S.), and US8148330, US9149444, US20150368324, US20150368325, US20150368326, and US20120328601 (B.S. and B.A.B.). This work was funded by an Edward R. and Anne G. Lefler Fellowship (S.H.), Coins for Alzheimer's Research Trust (B.S.), Fidelity Biosciences Research Initiative (F-Prime) (B.S. and C.A.L.), JPB Foundation (B.A.B.), the National Institutes of Health AG000222 (S.H.), National Institute of Neurological Disorders and Stroke-NIH R01NS083845 (D.J.S.), National Institute on Aging-NIH 1RF1AG051496A (B.S.). Supplementary materials contain additional data, including materials and methods. S.H. and B.S. designed the study and wrote the manuscript, with help from all authors. S.H. performed most experiments and data analysis; V.F.B .- G. and B.M.N. performed microglial activation and engulfment experiments along with immunohistochemistry; S.R. and K.M.M. performed C1q immunohistochemistry; A.F. performed FISH; S.L. performed electrophysiology; Q.S. and C.A.L. assisted with design and collection of APP/PS1 tissue; A.R. and B.A.B. designed and characterized the ANX-M1 anti-C1q antibody; and D.J.S. contributed in the discussions and experimental design. SUPPLEMENTARY MATERIALS www.sciencemag.org/content/352/6286/712/suppl/DC1 Materials and Methods Figs. S1 to S12 10 November 2015; accepted 18 March 2016 Published online 31 March 2016 10.1126/science.aad8373 716 6 MAY 2016 . VOL 352 ISSUE 6286 sciencemag.org SCIENCE Downloaded from http://science.sciencemag.org/ on January 2, 2020 Science Complement and microglia mediate early synapse loss in Alzheimer mouse models Soyon Hong, Victoria F. Beja-Glasser, Bianca M. Nfonoyim, Arnaud Frouin, Shaomin Li, Saranya Ramakrishnan, Katherine M. Merry, Qiaoqiao Shi, Arnon Rosenthal, Ben A. Barres, Cynthia A. Lemere, Dennis J. Selkoe and Beth Stevens Science 352 (6286), 712-716. DOI: 10.1126/science.aad8373originally published online March 31, 2016 Too much cleaning up The complement system and microglia seek out and destroy unwanted cellular debris for the peripheral immune system as well as excess synapses in the developing brain. Hong et al. now show how the system may go haywire in adults early in the progression toward Alzheimer's disease (AD). Aberrant synapse loss is an early feature of Alzheimer's and correlates with cognitive decline. In mice susceptible to AD, complement was associated with synapses, and microglial function was required for synapse loss. The authors speculate that aberrant activation of this "trash disposal" system underlies AD pathology. Science, this issue p. 712 ARTICLE TOOLS http://science.sciencemag.org/content/352/6286/712 SUPPLEMENTARY MATERIALS http://science.sciencemag.org/content/suppl/2016/03/30/science.aad8373.DC1 http://stm.sciencemag.org/content/scitransmed/7/309/309ra164.full RELATED CONTENT http://stm.sciencemag.org/content/scitransmed/6/241/241cm5.full http://stm.sciencemag.org/content/scitransmed/6/226/226ra30.full http://stke.sciencemag.org/content/sigtrans/8/402/ec329.abstract http://stke.sciencemag.org/content/sigtrans/5/238/ra61.full http://stke.sciencemag.org/content/sigtrans/8/373/ec100.abstract http://stke.sciencemag.org/content/sigtrans/9/427/ra47.full http://stke.sciencemag.org/content/sigtrans/9/427/pc11.full http://stke.sciencemag.org/content/sigtrans/10/470/eaan1468.full http://stm.sciencemag.org/content/scitransmed/7/278/278ra33.full REFERENCES This article cites 29 articles, 11 of which you can access for free http://science.sciencemag.org/content/352/6286/712#BIBL PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions Use of this article is subject to the Terms of Service Science (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. 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