complement c3 deficiency protects against neurodegeneration in … · complement c3 deficiency...

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

ALZHE IMER ’S D I S EASE

1AnnRomneyCenter forNeurologicDiseases, BrighamandWomen’sHospital, Building forTransformative Medicine, 9th Floor, 60 Fenwood Road, Boston, MA 02115, USA. 2HarvardMedical School, Boston,MA02115,USA. 3DepartmentofNeurology, F.M. KirbyNeurobiologyCenter, Boston Children’s Hospital, Center for Life Sciences, 12th Floor, 300 Longwood Ave-nue, Boston, MA 02115, USA. 4Harvard NeuroDiscovery Center NeuroBehavior Laboratory,Department of Neurology, Brigham and Women’s Hospital, Harvard Institute of Medicine,Room 945, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.*Corresponding author. Email: [email protected]

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Complement C3 deficiency protects againstneurodegeneration in aged plaque-rich APP/PS1 miceQiaoqiao Shi,1,2 Saba Chowdhury,1 Rong Ma,1 Kevin X. Le,1 Soyon Hong,2,3

Barbara J. Caldarone,2,4 Beth Stevens,2,3 Cynthia A. Lemere1,2*

The complement cascade not only is an innate immune response that enables removal of pathogens but also playsan important role in microglia-mediated synaptic refinement during brain development. Complement C3 is elevatedin Alzheimer’s disease (AD), colocalizing with neuritic plaques, and appears to contribute to clearance of Ab bymicroglia in the brain. Previously, we reported that C3-deficient C57BL/6 mice were protected against age-relatedand region-specific loss of hippocampal synapses and cognitive decline during normal aging. Furthermore, blockingcomplement and downstream iC3b/CR3 signaling rescued synapses from Ab-induced loss in young AD mice beforeamyloid plaques had accumulated. We assessed the effects of C3 deficiency in aged, plaque-rich APPswe/PS1dE9transgenic mice (APP/PS1;C3 KO). We examined the effects of C3 deficiency on cognition, Ab plaque deposition, andplaque-related neuropathology at later AD stages in these mice. We found that 16-month-old APP/PS1;C3 KO miceperformed better on a learning and memory task than did APP/PS1 mice, despite having more cerebral Ab plaques.Aged APP/PS1;C3 KO mice also had fewer microglia and astrocytes localized within the center of hippocampal Abplaques compared to APP/PS1 mice. Several proinflammatory cytokines in the brain were reduced in APP/PS1;C3 KOmice, consistent with an altered microglial phenotype. C3 deficiency also protected APP/PS1 mice against age-dependent loss of synapses and neurons. Our study suggests that complement C3 or downstream complementactivation fragments may play an important role in Ab plaque pathology, glial responses to plaques, and neuronaldysfunction in the brains of APP/PS1 mice.

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INTRODUCTIONThe complement cascade, of which C3 is the central molecule, plays apivotal role in the immune system. Upon activation, classical com-plement initiating protein C1q binds to and coats dead cells, debris,or pathogens, triggering a protease cascade, leading to the activation ofcomplement protein C3, which opsonizes material for elimination bytriggering phagocytosis of macrophages, or leading to the formation ofthe membrane attack complex, causing cell lysis (1). In addition, com-plement has been shown to play a role in microglia-mediated synapseelimination in the developing visual system to refine neuronal connec-tions (2–4). We recently demonstrated that C3 deficiency spared age-dependent synaptic and neuronal loss in a region-specific manner andprotected against cognitive impairment in normal aging of C57BL/6mice (5). Synapse loss occurs early in Alzheimer’s disease (AD)and correlates with cognitive decline (6, 7). Complement cascade com-ponents, including C3, are up-regulated and associated with amyloidplaques that typically contain dystrophic neurites in human AD(8, 9); however, the role of C3 in plaque-related synapse loss and neuro-degeneration inAD is unknown. Recently, we reported that in early-stageAD inmice, pre-plaque Ab oligomer–induced hippocampal synapse losswas rescued by both genetic deletion of complement and pharmaco-logical treatment with an anti-C1qa blocking antibody (10). However,it is not known whether blocking the complement cascade also protectsagainst cognitive impairment and neurodegeneration at later stages ofAD in mice with accumulation of amyloid plaques in the brain.

Several complement components, includingC1q, activatedC3 (C3b,C3c, and C3d), and C4 (8, 9, 11, 12), may be produced by glia

surrounding plaques in the AD brain. Ab peptides up-regulate C3 pro-duction by microglia and astrocytes in vitro (13, 14) and also increaseC3 expression and deposition in AD mouse brain in vivo (10, 15–18).Complement can recruit and activate microglia around fibrillar Ab de-posits (19) and mediates, in part, the uptake and clearance of Ab by theinteraction of complement component iC3b with its receptor CR3(CD11b/CD18) on the surface of microglia (20). We previously re-ported that aged C3-deficient J20 hAPP transgenic mice had a higherplaque load, slightly reduced neuron number, and altered macrophagepolarization compared to age-matched J20 mice (21). Others reportedthat C3 inhibition by expression of soluble complement receptor–relatedprotein y (sCrry) in J20 hAPP mice resulted in increased Ab accumu-lation, neuronal degeneration, and reducedmicroglia activation (22). Incontrast, C1q deficiency resulted in protection of synapses and neuro-pathology with decreased glial activation surrounding plaques in twoother AD amyloidosis models, Tg2576 and APP/PS1 mice (23). Inhibi-tion of C5a, a C3 downstream activation fragment, using a C5aR antag-onist resulted in rescue of a hippocampal-dependentmemory task (24).However, none of these previous reports addressed whether complementC3 plays a role in cognitive health. Hence, the consequences of C3 in theaging AD brain remain unclear.

Here, we generated APP/PS1;C3 KO to ask whether C3 plays a role inplaque-related neuropathological changes and cognitive decline.We demon-strate that C3 deficiency inAPP/PS1mice protected against age- and plaque-relatedsynapseandneuron loss,decreasedglial reactivity,andsparedcognitivedecline, despite an increased plaque burden in the mouse brain.

RESULTSSparing of cognitive decline in plaque-burdened aged APP/PS1;C3 KO miceAged APP/PS1 mice (>15 months of age) have been shown to exhibithyperactivity in the open-field test and impaired learning and spatialmemory in the radial arm/Morris water maze test compared with

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nontransgenic mice (25). To determine whether C3 deficiency affectsage-related cognitive changes in mice, we assessed learning andmemory in 16-month-old wild-type (WT), APP/PS1, APP/PS1;C3KO, and C3 KO mice. We used the water T-maze test, whichmeasured spatial learning and memory as the animals learned the lo-cation of a hidden platform (acquisition) and cognitive flexibility dur-ing reversal learning (26). Although APP/PS1 mice showed significantimpairment in finding the platform on acquisition day 2 compared toWT mice (P < 0.05; Fig. 1A), there were no significant differences inthe percent of mice that reached criterion (≥80% correct choices oneach individual day) between genotypes on day 6, indicating that allgroups eventually learned the task (Fig. 1A). Acquisition performance


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in APP/PS1;C3 KO mice was between that of WT mice and APP/PS1mice, indicating a nonsignificant trend, suggesting that APP/PS1;C3KO mice learned the location of the hidden platform more quicklythan did APP/PS1 mice. During the reversal trials, in which the plat-form location was switched, APP/PS1 mice demonstrated cognitiveimpairment by performing significantly worse than WT mice on days3 to 5 (Fig. 1A). Reversal learning in APP/PS1;C3 KOmice was similarto that of WT mice and C3 KO mice, and was significantly better thanthat of APP/PS1 mice on days 4 and 5 (Fig. 1A), suggesting that agedAPP/PS1;C3 KO mice had enhanced spatial learning and cognitiveflexibility compared to APP/PS1 mice.

The percent of mice that reached reversal criterion across all days(≥80% correct choices on two or more consecutive days) was signifi-cantly higher in WT C3 KO and APP/PS1;C3 KO mice compared toAPP/PS1mice (P < 0.001; Fig. 1B). Fewer APP/PS1mice reached rever-sal criterion compared to APP/PS1;C3KOmice, whereas C3KO, APP/PS1;C3KO, andWTmice achieved equivalent reversal criteria, suggest-ing that C3 deficiency in the APP/PS1 mice protected against both age-related and AD-related cognitive impairment.

Consistent with previous reports (25), we found that 16-month-oldAPP/PS1 mice traveled a significantly greater distance in the open-fieldtest compared toWTmice (fig. S1A).Overall, C3 deficiency inAPP/PS1mice had no effect on distance traveled in the open-field test (fig. S1A).We also conducted the elevated plus maze test to examine anxiolytic-like behavior in 16-month-old mice and found that APP/PS1;C3 KOmice showed greater anxiolytic-like behavior compared to APP/PS1mice (fig. S1, B and C) that was not due to differences in locomotoractivity (fig. S1D). These behavioral studies suggest that cognitive de-cline was at least partially spared in APP/PS1;C3 KO mice.

Elevated cerebral Ab plaque deposition and insoluble Ab inAPP/PS1;C3 KO miceTo determine whether memory improvement in APP/PS1;C3KOmicewas due to decreased Ab, we examined the Ab plaque load of 4- and16-month-old APP/PS1;C3KO and APP/PS1 mice. No significant dif-ferences in Ab plaque burden were observed between 4-month-oldAPP/PS1 andAPP/PS1;C3KOmice at the earliest stages of plaque dep-osition (fig. S2). However, by 16 months of age, Ab plaque depositionwas elevated in both the prefrontal cortex and hippocampus of APP/PS1;C3 KO mice compared to age-matched APP/PS1 mice (Fig. 2, Aand B, and fig. S3). In the prefrontal cortex, plaque deposition was sig-nificantly increased by 167.37% for Ab starting at the first N-terminalresidue (Ab1–x; detected by Ab N-terminal antibody, 3D6) (P < 0.01;Fig. 2A). In the hippocampus, plaque deposition was significantlyincreased by 63.4% for Ab ending at residue 42 (the longer, more path-ogenic form; Abx–42) (P < 0.01), by 64.1% for Ab ending at residue 40(the shorter, more abundant but less pathogenic form; Abx–40) (P <0.05), and by 65.4% forAb1–x (P< 0.05; Fig. 2B). Furthermore, we quan-tified the area of immunoreactivity for different sizes of Abx–42–labeledplaques. Larger plaques (>50 mm) were markedly increased by 127.0%(P < 0.01), whereas small (<20 mm) andmedium (20 to 50 mm) plaqueswere modestly but significantly increased by 28.7% (P < 0.05) and by56.0% (P < 0.01), respectively, in the hippocampus of APP/PS1;C3 KOmice compared toAPP/PS1mice (Fig. 2, C andD). Thioflavin S–positivestaining of fibrillar amyloid deposits was significantly elevated in thehippocampus of APP/PS1;C3 KO mice (P < 0.05; Fig. 2, C and E).

Enzyme-linked immunosorbent assay (ELISA) measuring Ab inmouse hemibrain homogenates revealed that guanidine-soluble (T-per–insoluble) Abx–40 and Abx–38 were significantly higher in APP/PS1;C3

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Fig. 1. APP/PS1;C3 KO mice show improved cognitive flexibility (reversal) com-pared to APP/PS1 mice at 16 months of age. (A) Percent of mice that reached crite-rion (≥80%correct choicesoneach individual day) in thewater T-maze test. Compared toWT mice, APP/PS1 mice were impaired in acquisition (day 2) and reversal learning andmemory (days 4 and 5) (*P < 0.05, **P < 0.01). APP/PS1;C3 KO mice performed signifi-cantly better thandid APP/PS1mice (##P < 0.01), but similar toWT andC3KOmice, in thereversal test on days 4 and 5, suggesting better cognitive flexibility in APP/PS1;C3 KOmice compared to APP/PS1mice. (B) In total, fewer APP/PS1mice reached the reversalcriterion (≥80% correct choices over two consecutive days) (*P < 0.05), whereas thepercent of WT, C3 KO, and APP/PS1;C3 KO mice that reached criterion in the reversaltest was higher compared to APP/PS1mice (***P < 0.001), indicating that C3 deficiencyin APP/PS1mice hadboth age-dependent andAD-related effects (WT,n=13; APP/PS1,n = 11; APP/PS1;C3 KO, n = 10; C3 KO, n = 11). Tests were assessed using one-wayanalysis of variance (ANOVA) followed by Fisher’s protected least significant differencepost hoc test.

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Fig. 2. IncreasedAb plaque load in16-month-oldAPP/PS1;C3KOmice. (A andB) Quantification of Abx–42, Abx–40, andAb1–x immunoreactivitieswithin a designated region ofinterest (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 < 0.01versus APP/PS1mice, independent unpaired t test per Ab species; n = 9mice; six equidistant planes, 150 mmapart). (C) Abx–42–immunoreactive and thioflavin S–positive plaqueswere higher in the hippocampus of C3-deficient APP/PS1 mice versus APP/PS1 mice. White circles indicate large plaques (>50 mm); black arrows indicate medium-sized plaques(>20 but <50 mm). Scale bars, 50 mm. (D) Quantification of small, medium, and large hippocampal plaques confirmed an increased plaque load in APP/PS1;C3 KOmice, especiallyfor 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 anincrease in fibrillar plaques in APP/PS1;C3 KOmice (*P < 0.05 versus APP/PS1 mice, unpaired t test; n = 6; three equidistant planes 300, mmapart). (F) Increased insoluble cerebralAbx–40 and Abx–38 were found in APP/PS1;C3 KO mice compared with APP/PS1 mice (*P < 0.05, independent unpaired t tests per Ab species; n = 8).

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KOmice compared to APP/PS1mice (P < 0.05; Fig. 2F). These data are inagreement with the corresponding increase in Abx–40 and Ab1–x plaqueload. These results indicate that the absence of a key complement factor,C3, or its downstream activation products in APP/PS1 transgenic micemight result in an age-dependent increase in the accumulation of cerebralAb plaques and insoluble Ab in aged APP/PS1 mice.

Altered plaque-associated gliosis in aged APP/PS1;C3KO miceMicroglia and astrocytes are frequently associated with plaques in ADbrain. Therefore, we examined colocalization of these cells with plaquesin mouse brain by immunostaining for Iba-1 and CD68 (markers ofmicroglia and macrophages) as well as glial fibrillary acidic protein(GFAP; amarker of astrocytes). Immunoreactivities to Iba-1 andGFAPwere similar between APP/PS1;C3 KO and APP/PS1 mice at postnatalday 30 (P30) and 4 months of age (fig. S4). However, 16-month-oldAPP/PS1 transgenic mice displayed microglia and macrophages withenlarged cell bodies and thicker, shorter processes with less branchingcompared to Iba-1–positive andCD68-positive cells inAPP/PS1;C3KOmice (Fig. 3A and fig. S5, A and B); this was consistent with morpho-logical changes associated with reactive microgliosis. In addition,GFAP-positive astrocytes were clustered in hippocampus of APP/PS1mice butweremorewidely distributed and less clustered aroundplaquesin APP/PS1;C3KOmice (Fig. 3A). Quantitative image analysis revealedthat Iba-1 immunoreactivity (% area) was significantly reduced by28.8% in the hippocampal CA3 region (P < 0.05) and slightly reducedby 19.3% (nonsignificant trend) in the hippocampal CA1 region inAPP/PS1;C3 KO mice; no significant differences were seen in the den-tate gyrus (Fig. 3B). In addition, CD68 immunoreactivity, indicative oflysosomal phagosomes, was significantly reduced in hippocampal CA3,CA1, and dentate gyrus in APP/PS1;C3 KO mice (P < 0.05 per region;Fig. 3C). GFAP immunoreactivity of astrocytes in CA3, CA1, and den-tate gyrus was also significantly reduced in APP/PS1;C3 KO mice (P <0.05, CA1; P < 0.01, CA3 and dentate gyrus; Fig. 3D). Further, immu-nofluorescence intensities for Iba-1 and CD68 in Ab plaques of similarsize were significantly reduced in hippocampal CA3 of 16-month-oldAPP/PS1;C3 KO mice compared to APP/PS1 mice (P < 0.01; Fig. 3, Eand F). Although glial cell morphology was different, stereological anal-ysis revealed that the number of Iba-1– and GFAP-positive cells in hip-pocampal CA3, CA1, and dentate gyrus areas was not significantlydifferent between aged APP/PS1;C3 KO mice and APP/PS1 mice(Fig. 3, G and H).

Next, glial cell association within and surrounding Ab plaques wasexamined to further elucidate a possible mechanism underlying the C3deficiency–mediated increase in hippocampal Ab plaque burden inAPP/PS1;C3 KO mice. We quantified the immunofluorescence inten-sity and the number of microglia/macrophages (Iba-1–positive) andastrocytes (GFAP-positive) surrounding 6E10 antibody–positive Abplaques of similar size in the hippocampal CA3 region of 16-month-old APP/PS1;C3 KO mice and APP/PS1 mice using high-resolutionZ-stack imaging by confocal microscopy (Fig. 4, A to L). Orthogonalanalysis revealed that Iba-1–positive cells and Ab plaques colocalized(fig. S5A). Three-dimensional (3D) reconstructed imageswere analyzedfor intensity and cell number in the proximal area (within the center oflarge plaques) and distal area (surrounding large plaques).We observedmore Iba-1– and GFAP-positive cells in the center of plaques in APP/PS1 mice, whereas Iba-1– and GFAP-positive cells tended to surroundthe plaques in APP/PS1;C3 KO mice (Fig. 4, A and G) and were morewidely distributed. Upon quantification, we found that the intensity of


Iba-1–positive cells in APP/PS1;C3 KO mice was significantly reducedproximal to plaques (P < 0.01) and significantly increased distal to pla-ques (P < 0.01) compared to APP/PS1mice (Fig. 4, C andD). Similarly,the intensity of GFAP-positive astrocytes in APP/PS1;C3 KOmice wassignificantly reduced in the proximal area of plaques (P < 0.05) and sig-nificantly increased distal to plaques (P < 0.01) compared to APP/PS1mice (Fig. 4, I and J). Although the number of glial cells located withinplaques (proximal) was decreased (Fig. 4, E andK) and those surroundingplaques (distal) were increased (Fig. 4, F and I) in APP/PS1;C3 KOmice,there was no difference in the total number of glial cells in hippocampalCA3 in APP/PS1;C3KO versus APP/PS1 mice (Fig. 3, G and H). Thus,although the APP/PS1;C3 KO mice had enhanced plaque deposition,they also had reduced immunofluorescence intensity and localizationof glia within the center of plaques, suggesting that C3 deficiencymay alter the glial response to plaques.

In a previous study, we found that CD45 immunoreactivity wasincreased in aged J20;C3 KO mice compared to J20 hAPP trans-genic mice. However, Iba-1 immunoreactivity and the amount ofCD68 by Western blot were not significantly different betweenJ20;C3 KO and J20 mice (15). Here, we performed immunostainingof Iba-1 and CD68 on brains from 18-month-old APP/PS1, APP/PS1;C3 KO, J20, and J20;C3 KO mice. In aged J20 mice, both Iba-1 and CD68 immunoreactivities within Ab plaques were lower thanthose in aged APP/PS1 mice (fig. S6, A and D). We observed de-creases in Iba-1 and CD68 immunoreactivities within Ab plaquesin APP/PS1;C3 KO mice compared to APP/PS1 mice (fig. S6, E andG) but no difference between J20 mice and J20;C3 KO mice (fig. S6,F and G), consistent with our previous study (16).

Altered cytokines in aged APP/PS1;C3 KO mouse brainELISAof soluble proteins derived fromT-per extraction of brain homo-genates revealed that proinflammatory cytokines including TNF-a,IFN-g, and IL-12p70, which have been reported to be elevated in ADbrain (27), were significantly reduced in 16-month-oldAPP/PS1;C3KOmice compared toAPP/PS1mice (P<0.05; Fig. 4M). The amount of IL-10did not differ between APP/PS1 and APP/PS1;C3 KOmice, although theratio of IL-10/IL-12was significantly increased inAPP/PS1;C3KOmicecompared to APP/PS1 mice (P < 0.05; Fig. 4M). Thus, C3 deficiencyresulted in the reduction of several proinflammatory cytokines in agedAD mice.

Partial protection against age-related hippocampal synapticdegeneration in APP/PS1;C3 KO miceDecreased synaptic number, mRNA, and protein as well as impairedsynaptic morphology have been reported in hippocampus and cortexof APP/PS1 mice between 7 and 18 months of age (28–30). To deter-mine the effects of C3 on hippocampal synapses in aged APP/PS1mice,we performed high-resolution confocal microscopy analysis of pre- andpostsynaptic markers (that is, synaptic puncta) in hippocampal CA3,CA1, and dentate gyrus areas in 16-month-old mice and comparedWT to C3 KO, WT to APP/PS1, APP/PS1 to APP/PS1;C3 KO, andC3 KO to APP/PS1;C3 KO mice (Fig. 5). Consistent with our previousresults (5), C3 KO mice had significantly more Vglut2-positive (P <0.01), GluR1-positive (P < 0.05), and colocalized (P < 0.05) synapticpuncta than WT mice (Fig. 5, A to C). In addition, C3 KO mice hadmore presynaptic Vglut2-positive puncta and more postsynapticGluR1-positive puncta than APP/PS1 and APP/PS1;C3 KO mice (P <0.01), although colocalized synaptic punctawere not significantly differ-ent between C3 KO and APP/PS1;C3 KO mice (Fig. 5, A to C). We

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Fig. 3. Morphological changesassociatedwith glial activationwere reduced in 16-month-oldAPP/PS1;C3KOmice. (A) Iba-1–and CD68-positive immunos-taining showed less activationofmicroglia andmacrophages(that is, smaller cells with thin-nerprocesses)and lessclusteringof GFAP-positive astrocytes inthe hippocampal CA3 region of16-month-old APP/PS1;C3 KOmice compared to APP/PS1mice. Scale bar, 50 mm. (B to D)Quantification of Iba-1, CD68,and GFAP immunostaining inhippocampal CA3, CA1, anddentategyrus (DG) showed re-duced glial immunoreactivity(IR) inAPP/PS1;C3KOmice com-pared to APP/PS1 mice (*P <0.05, **P < 0.01, independentunpaired t tests per region; n =6 to 8; three equidistant planes,300mmapart). (E)High-resolutionconfocal images of Ab plaquesimmunostained with 6E10 anti-body show microglia/macro-phages (immunoreactive forIba-1) and phagocytic cells (im-munoreactive for CD68) in thehippocampalCA3 region. Thesefindings suggested reducedphagocytosis in APP/PS1;C3 KOmice compared to APP/PS1mice. Scale bar, 10 mm. DAPI,4′,6-diamidino-2-phenylindole.(F) Iba-1– and CD68-positiveimmunofluorescence intensi-ties were lower in APP/PS1;C3KO mice compared to APP/PS1mice (**P < 0.01, independentunpaired t tests per marker;n = 5). (G and H) Stereologicalcounts of Iba-1–immunoreactivecells (G) and GFAP-immuno-reactive cells (H) counterstainedwith 3,3′-diaminobenzidinewereperformed inhippocampalCA3, CA1, and dentate gyrustissue. The number of Iba-1–positivemicroglia/macrophageswas increased in CA3 and den-tate gyrus in APP/PS1 and APP/PS1;C3 KO mice versus WT andC3 KO mice (G). The number ofGFAP-positive astrocytes wasincreased only in the hippocam-pal CA3 region (H); no differ-
nces 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; threequidistant planes, 300 mm apart).
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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, andH) High-resolutionconfocal imagesof Iba-1 (red)/6E10 (Ab antibody; green)/DAPI (blue)orGFAP (red)/6E10 (green)/DAPI (blue) inAPP/PS1andAPP/PS1;C3KOmice. The inner ring indicates theproximal regionof aplaque (that is, the center), whereas the outer ring indicates the distal region. Scale bars, 10 mm. (C,D, I, and J) Immunofluorescence intensities of Iba-1 and GFAP were lower in the Ab plaqueproximalarea(CandI)andhigher intheAb plaquedistalarea (DandJ) inAPP/PS1;C3KOmicecomparedtoAPP/PS1mice(*P<0.05, **P<0.01,unpaired t test;n=6). (E,F,K, andL)ThenumberofIba-1–andGFAP-positivecellswas reduced in theproximalplaquearea inAPP/PS1;C3KOmicecompared toAPP/PS1mice (EandK) (*P<0.05)and increased in thedistalplaquearea (FandL) (*P<0.05, unpaired t test;n=6). (M) Assayof cytokinesbyELISA inmousebrainhomogenates revealed reductions in tumornecrosis factor–a (TNF-a), interferon-g (IFN-g), and interleukin-12 (IL-12) andan increase in the IL-10/IL-12 ratio in16-month-oldAPP/PS1;C3KOmice compared toAPP/PS1mice (*P<0.05, independentunpaired t test permarker followedbyBonferronicorrection for multiple comparisons; n = 8). KC-GRO, keratinocyte chemoattractant (KC) chemokines CXCL1/2, mouse homologues of human growth-regulated oncogenes (GRO).




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Fig. 5. C3deficiency resulted inpartial pres-ervation of synapse density in APP/PS1micedespite an increased plaque load. Com-parisons were made between WT and C3 KO,WT and APP/PS1, APP/PS1 and APP/PS1;C3 KO,andC3KOandAPP/PS1;C3KOmice. (A) Synap-tic puncta of pre- and postsynaptic markersVglut2 and GluR1, respectively, and their colo-calization in hippocampal CA3 were analyzedby high-resolution confocal microscopy in16-month-old mice. Scale bar, 5 mm. (B) C3KO mice had increased Vglut2 and GluR1 syn-aptic densities compared to WT, APP/PS1, andAPP/PS1;C3 KO mice. APP/PS1 mice had fewerGluR1 synaptic densities than WT mice,whereas APP/PS1;C3 KOmice had more GluR1densities than APP/PS1 mice and were notsignificantly different from WT mice (*P <0.05, **P< 0.01, one-wayANOVA andBonferro-ni post hoc test per marker; n = 6 to 8; threeequidistant planes, 300 mm apart). (C) Colocali-zation of pre- and postsynaptic puncta re-vealed increased puncta in C3 KO versus WTand APP/PS1 but not APP/PS1;C3 KO mice, re-ducedpuncta inAPP/PS1mice versusWTmice,and a rescue of synaptic puncta in APP/PS1;C3KO mice compared to APP/PS1 mice, suggest-ing partial protection against synapse loss bydeletion of C3 (one-way ANOVA and Bonferro-ni post hoc test). (D and E) Western blotting ofsynaptic proteins in hippocampal synapto-somes isolated from aged mice indicatedincreased postsynaptic proteins GluR1,PSD95, and Homer1 in C3 KO versus WT,APP/PS1, and APP/PS1;C3 KO mice. APP/PS1mice had lower postsynaptic GluR1, PSD95,and Homer1 and presynaptic SYN-1 and SYPcompared to WT mice. APP/PS1;C3 KO micehad more GluR1, PSD95, Homer1, SYN-1, andSYP than APP/PS1 mice and were not signifi-cantly different than WT mice, suggesting asparing of synaptic loss in aged C3-deficientAPP/PS1 mice (*P < 0.05, **P < 0.01, one-wayANOVA and Bonferroni post hoc test permarker; n = 6). (F and G) Western blottingand quantification of TrkB, mBDNF, CREB, andpCREB in hippocampal homogenates of16-month-old mice. C3 KOmice had increasedmBDNF and pCREB compared to WT, APP/PS1,and APP/PS1;C3 KO mice. APP/PS1 miceshowed reductions in all four markers com-pared to WT mice. APP/PS1;C3 KO mice hadhigher mBDNF, CREB, and pCREB than APP/PS1mice (*P < 0.05, **P < 0.01, one-way ANOVA andBonferroni post hoc test permarker;n=6), sug-gesting a partial rescue of age- and AD-relatedlowering of BDNF pathway proteins. (H) Tablesummarizing the effects of C3 deficiency onsynaptic and BDNF-related proteins. N.S., notsignificant.

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found that the densities of postsynaptic puncta (GluR1, P < 0.01) andcolocalized puncta (Vglut2 and GluR1, P < 0.05) were significantly re-duced in hippocampal CA3 in 16-month-old APP/PS1 mice comparedto WT mice (Fig. 5, A to C). The density of GluR1-positive and colo-calized synaptic puncta was significantly increased in CA3 of APP/PS1;C3 KOmice compared to APP/PS1 mice (P < 0.01) and normalized toWT, suggesting protection against age-related synapse loss (Fig. 5H).Western blotting of presynaptic markers, synapsin-1 (SYN-1) and sy-naptophysin (SYP), andpostsynapticmarkers,GluR1,PSD95, andHomer1,in hippocampal synaptosomes from 16-month-oldWT,C3KO, APP/PS1,and APP/PS1;C3 KO mice revealed that C3 KO mice had elevatedGluR1, PSD95, and Homer1 compared to all other groups, and APP/PS1micehadreducedGluR1,PSD95,Homer1, SYN-1, andSYPcomparedtoWTmice (Fig. 5, D, E, and H). APP/PS1;C3 KOmice showed a signif-icant increase in all five synaptic proteins compared to APP/PS1mice (P <0.05, Syn-1; P < 0.01, GluR1, PSD95, Homer1, and SYP), and the amountswere similar to those detected in WT mice (Fig. 5, D and E). In addition,microtubule-associated protein 2 (MAP-2), a marker of the neuronal cellbody and dendrites, was more abundant in fibrillar, thioflavin S–positiveamyloid plaques of similar size in the hippocampal CA3 region of APP/PS1;C3 KO mice compared to APP/PS1 mice (P < 0.01; fig. S7).

Mature brain-derived neurotrophic factor (mBDNF) and its up-stream (TrkB) and downstream [CREB (cyclic adenosine monopho-sphate response element–binding protein) and pCREB] signalingpartners, key mediators of synaptic plasticity and memory, wereexamined by Western blot of brain homogenates and quantified(Fig. 5, F to H). C3 KO mice had higher mBDNF and pCREB thanWT, APP/PS1, and APP/PS1;C3 KO mice. APP/PS1 mice had reducedTrkB, mBDNF, CREB, and pCREB compared to WT mice. APP/PS1;C3 KO mice had significantly higher mBDNF, CREB, and pCREBcompared to APP/PS1 mice (P < 0.01) but lower mBDNF and pCREBthan C3 KO mice (Fig. 5, F and G). No significant differences wereseen in CREB between C3 KO and APP/PS1;C3 KO mice. This resultcorrelates well with our cognitive and synaptic puncta data and sug-gests a pro-cognitive phenotype in the APP/PS1;C3 KO mice. Thus,whereas 16-month-old APP/PS1 mice demonstrated hippocampalCA3 synapse loss, APP/PS1;C3 KO mice were at least partially sparedsynapse loss, which was associated with an increase in BDNF signalingpathway proteins (Fig. 5). We also examined the amounts of humanAPP and presenilin 1 in APP/PS1 and APP/PS1;C3 KO mice andfound no significant differences in either protein between the two gen-otypes (fig. S8).

Absence of age-dependent hippocampal CA3 neuron loss inAPP/PS1;C3 KO miceAs previously reported (5), there was a small (28%) but statistically sig-nificant age-dependent reduction in the number of NeuN-positive neu-rons in the hippocampal CA3 region from P30 to 16 months of age inWTmice that was rescued inC3KOmice (P< 0.05; Fig. 6F). A stronger(40%) reduction in CA3 neurons was seen from P30 to 16 months ofage in APP/PS1 mice (P < 0.01; Fig. 6F). APP/PS1;C3 KO and C3 KOmice showed no significant age- or plaque-related loss of hippocampalCA3 neurons (Fig. 6F). Instead, 16-month-old APP/PS1;C3 KO micehad significantly more hippocampal CA3 neurons compared to age-matched APP/PS1 mice (P < 0.05; Fig. 6, A and B). No differences inneuron numberswere found inCA1, dentate gyrus, and prefrontal cortexareas inAPP/PS1;C3KOmice compared toAPP/PS1mice (Fig. 6, A andC to E). In addition, CA3 neurons were disorganized in 16-month-oldAPP/PS1 mice but not in APP/PS1;C3 KO mice (Fig. 6A). These data


suggest that hippocampal CA3 neurons are more vulnerable to agingthan neurons in other brain regions in both WT and APP/PS1 miceand that APP/PS1;C3KOmice may be protected against this age-relatedneuronal vulnerability even in the presence of abundant plaques.

DISCUSSIONComplement C3 is an innate immune system molecule that is impor-tant for removing pathogens and eliminating synapses during brain de-velopment and aging (2–5). Complement activation is elevated inhuman AD brain, especially in the presence of fibrillar amyloid plaquescontaining clusters of reactivemicroglia (8, 9, 11, 12) and in the brains ofAD amyloidosis mouse models (10, 17, 31). To address the role ofcomplement C3 in age-dependent, plaque-related neuropathology, wegenerated APP/PS1;C3 KO mice and analyzed the role of C3 in brainbiochemistry, neuropathology, and behavior in 16-month-oldmice.Wedemonstrated that C3 deficiency protected against cognitive impair-ment, loss of hippocampal CA3 synapses and neurons, and glial altera-tions even in the context of increased Ab plaque deposition. Our resultssuggest that, despite extensive plaque deposition, C3 deficiency protectsagainst both age- and AD-related synapse loss and cognitive decline inaged APP/PS1mice by altering the glial response to plaques. These dataindicate that plaque load is less critical than the reaction of glia to theplaques, and implicate complement activation as a possible link betweenfibrillar amyloid and accompanying microglial clustering in late-stageAD pathogenesis in mice, similar to that observed in human AD brain.

Progressive cognitive impairment in water maze tests is observed inaged APPswe/PS1dE9 mice (an AD-like mouse model of amyloidosis)and is correlated with progressive Ab deposition, neuroinflammation,and degeneration of synapses and neurons (25, 32). Here, we foundthat APP/PS1;C3 KO mice were protected against cognitive deficits,especially during water T-maze test reversal learning, a task that ishippocampal-dependent (20). This result agrees with previous reports,including our own, demonstrating that C3 KO mice are protectedagainst synapse and neuron loss and are spared from cognitive impair-ment during normal aging (5, 33). Many of our results in the currentstudy point to age-dependent protection by C3 deficiency in agedAPP/PS1 mice. However, C3 deficiency also protected against someAD-related changes because there were no significant differences be-tween APP/PS1;C3 KO, WT, and C3 KO mice in the percent of micethat reached the reversal criterion in the water T-maze test, indicatingbetter cognitive flexibility. Similarly, C3 deficiency protected againstAD-related decline in hippocampal SYP and CREB expression inAPP/PS1 mice. In addition, inhibition of complement C5aR, down-stream of C3 activation, with an antagonist has been reported to re-duce Ab pathology and improve cognitive function in transgenic 2576AD mice (24), raising the possibility that the protective cognitiveeffects we observed in aged C3-deficient APP/PS1 mice might bedue to the lack of downstream C5a/C5aR signaling. Synaptic degen-eration has been correlated with cognitive decline in AD (6, 7); there-fore, our observations of cognitive sparing in aged APP/PS1;C3 KOmice may be due to the preservation of hippocampal synapses andneurons, even in the presence of increased plaque load. Whether theprotective effects of C3 deficiency overpower the toxic effects of Ab orcause sequestration of Ab into plaques remains to be determined.Neuroinflammation (for example, glial clustering and activationwith-in plaques) was reduced in APP/PS1;C3 KOmice, suggesting that C3may play a critical role in driving neuroinflammation, which facilitatessynaptic decline.

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16 monthsFig. 6. C3 deficiency resulted in partial sparing of neuron loss in hippo-campal CA3 in 16-month-old APP/PS1 mice. (A) NeuN immunoreactivity inhippocampal CA3, CA1, and dentate gyrus (DG) and prefrontal cortex (PFC)regions in APP/PS1 andAPP/PS1;C3KOmice. Scale bars, 50 mm. (B) APP/PS1;C3KO 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 inneuron numbers in CA1, dentate gyrus, and PFC between APP/PS1 and APP/PS1;C3KOmice. (F)Age-dependentneuron losswasobservedbetweenP30and16months of age inWTandAPP/PS1miceandalsobetween4and16monthsofage inAPP/PS1mice. However, neuron losswasnot observed inC3KOorAPP/PS1;C3KOmice (*P<0.05, **P<0.01, two-wayANOVAandBonferroniposthoctest; n = 6 to 8; six equidistant planes, 150 mm apart).





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In the brain environment undergoing injury or disease, chronic ac-tivation of microglia may contribute to synaptic degeneration andmemory decline (34). Elimination of microglia and inhibition of mi-croglial proliferation by inhibiting colony-stimulating factor 1 receptorhave been reported to prevent spine and neuronal loss and improvememory performance in AD mouse models, without significant mod-ulation of Ab pathology (35, 36). In addition, we recently demonstratedthat complementandmicrogliamediate synapse loss inADmousemodelsat early AD stages before plaque deposition (10). Our data here demon-strate that complement plays an additional role in the glial response toamyloid deposition and synaptic health at later AD stages as well. Further-more, early C1q-mediated microglial activation and neurodegenerativechanges in retina observed in a mouse model of retinal ischemia and re-perfusion were abrogated in C1qa KO mice (37). Similarly, the loss ofretinal ganglion cell synapses and dendrites (which precede axon and so-ma loss) in a genetic mouse model (DBA/2J) and an inducible rat model(microbead) of glaucomawere rescued by genetic deletion ofC1qa in themouse model and C1 inhibition in the rat model (38). Together, thesefindings suggest that inhibition of complement and its interaction withmicroglia may serve as a target to prevent the progression of synapse andneuron loss in AD.

Complement C3 plays a pivotal role in plaque deposition and clear-ance (20); however, it is not known whether C3 mediates the glial re-sponse in plaque-enriched brain. Here, we show that C3 deficiency inagedAPP/PS1mice decreasedmicroglial CD68 activity,maintainedmi-croglial branching, and increased plaque load, while preserving cogni-tion and hippocampal CA3 synapses and neurons. Both C3 deficiencyand inhibition of C3 convertase by overexpression of sCrry in J20 hAPPtransgenicmice led to an age-dependent increase in plaqueburden (21,22).In agreement with these studies in J20mice, we found that aged APP/PS1;C3 KO mice had enhanced plaque deposition and increased insolubleAb in brain homogenates. Microglial uptake and clearance of Ab (thatis, by phagocytosis) may be mediated by the interaction of iC3b with itsreceptor, CR3 (CD11b/CD18), on the surface of microglia (20, 39). Pre-vious studies have reported the reduction of microglial marker F4/80,I-A/I-E, or CD68 inC1q-deficient transgenic 2576 APPmice (23), J20hAPP/sCrry mice (22), and J20 hAPP;C3KOmice (21). Here, we alsofound that microglia/macrophage and astrocyte markers were re-duced in aged APP/PS1;C3 KO mice compared to APP/PS1 mice, al-though there was no difference in glial cell number. Moreover, we foundfewermicroglia and astrocytes located within plaques, suggesting that C3deficiency alters the glial response to plaques in aged, plaque-rich APP/PS1;C3 KOmice. Thus, these findings suggest that the increased fibrillarAb plaque load in the aged APP/PS1;C3KOmice is due to a muted glialresponse and reduced phagocytosis of Ab.

Senile plaques have been proposed to act as a potential reservoir of sol-uble oligomeric Ab. When nonsequestered, Ab colocalizes with the post-synapticdensity, is associatedwithdendritic spine collapse, and is themajorsynaptotoxic Ab species in the AD brain (40). Therefore, in theory, it ispossible that althoughC3deficiency reducedmicroglia-mediated phagocy-tosis of Ab and increased plaque deposition in APP/PS1 mice, it also mayhave resulted in more sequestration of Ab oligomers into plaques. Thismight reduce the availability of Ab oligomers to bind to synapses, therebyblockingmicroglia activation–induced synapse loss in the hippocampus inaged APP/PS1;C3 KOmice. This possibility would be consistent with ourprevious findings that genetic deletion or antibody blocking of C1qaprotected hippocampal synapses from Ab oligomer–induced damage(8). Although purely speculative at this stage, future studies are underway to further clarify this possibility.


Complement C3 may contribute to region-specific and age-dependent synapse and neuron loss in AD mice via C3-dependentchronicmicroglial activation. Complement is involved in synaptic elim-ination in brain development and aging. Upon activation, complementmediates synapse elimination in the developing visual system bycomplement tagging of synapses that are then phagocytosed bymicrog-lia (2, 3). Complement C3 contributes to age-dependent and region-specific synapse and neuron loss during normal aging (5). Synapse lossis an early and important change in AD brain (41), and hippocampalsynapse loss is observed in APP/PS1 mice (28, 29). Although bothplaque-associated and plaque-independent hippocampal synaptic de-generation have been observed in aged APP/PS1 mice (42), we foundreduced synaptic degeneration despite increased plaque load in agedAPP/PS1;C3 KO mice compared to APP/PS1 mice, indicating thatthe presence of plaques alone did not induce neurodegenerativechanges. Given that microglia and astrocytes, both of which expresscomplement, are highly phagocytic and participate in synapse pruning(10, 43), our findings that C3 deficiency protected against hippocampalsynaptic decline in two mouse models (C3 KO and APP/PS1;C3 KO)suggest that complement C3 or its downstream activation fragments,such as C3a, C5a, and C5b-9, may play an important role in synapseloss and neurodegeneration. Other reports support this hypothesis.For example, a recent report suggests that complement C3a secretionby astrocytes and binding to C3aR on microglia modulate Ab in vitroand amyloid plaques in vivo inADmousemodels (44). The same grouppreviously demonstrated that Ab-induced nuclear factor kB–mediatedrelease of C3a from astrocytes bound to C3aR on neurons and inducedneurodegeneration and cognitive impairment, whereas treatment witha C3aR antagonist rescued these effects (14). Others have reported thatlow-dose treatment with the same C3aR antagonist in an ischemia/reperfusion strokemodel in C57BL/6mice resulted in enhanced neuro-genesis, histologic and functional neuroprotection, and an anti-inflammatory effect (for example, the absence of T lymphocytesexpressing C3aRmigrating into the ischemic region) 7 days after injury(45). However, several caveats of the C3aR antagonist includingpotential off-target and agonist activity have been reported (46), leavingthe role of C3a/C3aR signaling in neurodegeneration unclear.

Previously, we reported an age-dependent loss of CA3 neurons in16-month-old C57BL/6 mice (5). Age-dependent neuron loss inAPPswe/PS1dE9 mice has been found in striatum (47) but not cortex(48) or hippocampal CA1 (30). Here, we report age-dependent loss ofhippocampal CA3 neurons in the same APP/PS1 mouse model, whichwas reduced in the absence of complement C3. Age-dependent neuronloss was not observed in CA1, dentate gyrus, and prefrontal cortex inaged APP/PS1;C3 KO mice, indicating that certain brain regions aremore vulnerable to neuron loss with aging than others. It is possible thatthe selective vulnerability of CA3 neurons during aging in APP/PS1mice is due to earlyCA3 synapse loss preceding late neuron loss inducedby C3-mediated synaptic elimination, similar to what we found in agedWT mice (5). CA3 neurons receive input from other CA3 neuronsthrough recurrent connections as well as medial septum and the diag-onal band of Broca, both of which show degenerative changes duringaging (49, 50). C1qa-deficient transgenic 2576 hAPP mice showincreased iC3b deposition in brain, reduced gliosis and neuron loss inseveral brain regions, and partial protection of synapses in the hippo-campal CA3 region, suggesting selective regional vulnerability ofsynapses and differential effects of various complement pathways (23).

It has been shown that microglia engulf presynaptic inputs via theC3/CR3-dependentmicroglia phagocytic signaling pathway in the early

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developing postnatal rodent brain (3). Recently,Hong et al. reportedAboligomer–induced, complement-mediated, and microglia-dependentsynaptic loss in young, pre-plaque AD mouse brain that was rescuedby the genetic deletion of C1q or C3, or by treatment with a C1qablocking antibody (10). This study suggests a role for complement inoligomeric Ab–induced synaptic pathology at early AD stages beforeplaque deposition. Here, we demonstrate a role for complement C3signaling in Ab fibril–induced clustering and activation of microgliawithin plaques at a much later disease stage in aged APP/PS1 mice.Ab-induced complement activation is strongly determined by the de-gree of Ab fibrillarity (51, 52). In humans, early, nonfibrillar diffuse pla-ques are associated with little or no microglial clustering (53) andaccumulate little or no complement proteins (8, 9). Complement hasbeen shown to promote the nucleation phase of Ab aggregation (54),suggesting that, once activated, complementmay perpetuate ADpatho-genesis. At later stages of AD pathogenesis in humans, variouscomplement proteins and clusters of activated microglia have beenfound to colocalize with fibrillar amyloid plaques, which often containdystrophic neurites (8, 9). Our current study supports the role ofcomplement in the link between Ab fibrillarity and clustering of acti-vated microglia and shows that lifelong C3 deficiency suppresses theglial response to plaques, reduces the production of some proinflamma-tory cytokines, rescues hippocampal synapses and neurons, and sparescognition in aged APP/PS1 mice despite increased plaque deposition.

Whether microglia and complement work together to mediate syn-apse loss in plaque-rich AD brain is unclear, but this hypothesis issupported by our data in APP/PS1 mice. Others have suggested thatC3-dependent microglia overactivation is responsible for neuroinflam-mation, which accelerates pathogenesis in neurodegenerative diseases(55). For instance, lipopolysaccharide-induced loss of dopaminergicneurons was inhibited by C3 deficiency (56). In experimental auto-immune encephalomyelitis, a mouse model of human multiple sclero-sis, C3 overactivation in Crry-deficient mice increased microglialoveractivation and exacerbated neurodegeneration (57). Mice deficientfor complement receptor 2 (Cr2 KO), which lack the CR1 and CR2 re-ceptors that bind to C3 activation fragments, had reduced secondarybrain damage after closed head injury including decreased neurondeath, astrocytosis and microglial activation, and improved neurologi-cal outcomes (58). In addition, genetic deletion of C3 (but not C1q)protected against synapse loss and promoted faster recovery 1 week af-ter axotomy of spinal cord motor neurons by sciatic nerve transection(59). Furthermore, mice deficient in BDNF in microglia have deficits inlearning andhippocampal synapse formation, indicating thatmicroglialBDNF signaling plays an important role in hippocampal function (60).Together, these results provide evidence for a deleterious effect ofcomplement activation and its downstream consequences in brain inthe context of aging, trauma, and neurodegenerative diseases.

Seemingly contradictory results of complement have been observedamong different AD mouse models. C3 inhibition enhanced plaquedeposition and promoted neurodegeneration in J20 hAPP mice eitherby overexpression of sCrry (22) or by genetic deletion of C3 (21). Theaddition of a mutant human PS1 transgene is one possible explanationfor the difference in C3 deficiency between J20mice in the previous stu-dies and APP/PS1 mice in this study. Previous reports demonstratedup-regulation of C1q in the hippocampus and GFAP and cathepsin Sin the cortex and hippocampus in conditional PS1/2 double-knockoutmice and increased expression of complement cascade componentsC1q,C3aR, andC4b in the hippocampi of PS1/2 double-knockoutmice(61, 62). Thus, it is possible that the presence of the mutant human PS1


gene in the APP/PS1 mice may explain, in part, the differential effect ofC3 deficiency on neurodegeneration compared to J20 hAPP mice.However, we found no significant difference in the amount of APPor PS1 in the APP/PS1 versus APP/PS1;C3 KO mice used here. Otherstudies demonstrated that lifelong C1q deficiency rescued SYP andMAP-2 expression in hippocampal CA3 in aged transgenic 2576APP and APP/PS1 mice (23), similar to our findings in 16-month-old APP/PS1;C3 KO mice. However, an additional study suggests thatC1q does not appear to play a role in synapse loss during aging but in-stead contributes to a decline in functional brain wiring (31). Here, weobserved less reactive microgliosis and reduced astrocytosis in agedAPP/PS1;C3KOmice, compared to no change in Iba-1 andCD68mar-kers in J20;C3 KOmice, similar to our previous study. In addition, ourcurrent findings showing hippocampal region–specific and glia/synapsemarker–specific effects described in APP/PS1;C3 KO mice may havebeen missed in our previous J20;C3 KO study that used fewer markersand examined the entire hippocampus, not individual subregions.Together, the differences in baseline glial responses to plaques andeffects of C3 deletion on microglia in aged J20 versus aged APP/PS1mice may explain the differences in neurodegeneration that we haveobserved in the two models.

Last, we acknowledge the limitations of our study, including theoverexpression of human mutant APP and PS1 in mice, which likelygenerates other APP fragments that could affect behavior. Our findingsare also limited by the differences in the immune system and life spanbetween mice and humans. In addition, our APP/PS1 mouse modellacked tau pathology (which better correlates with cognitive declinein human AD), thus limiting the translation of our findings to the fullhuman disease. Future studies in APP knockin mice or mice that haveboth amyloid and tau pathologies (for example, 3xTg-ADmice) wouldbe useful to confirm the protective effects of C3 deficiency that we ob-served in our aged APP/PS1 mice, and these other models might betterreflect human AD.

In summary, we found that C3 deficiency in APP/PS1 mice confersneuroprotection against age- and AD-related neurodegeneration andcognitive decline despite enhancing plaque burden. Our results suggestthat C3 may play a detrimental role in synaptic and neuronal functionin plaque-rich, aged brain and suggest that inhibition of C3 signalingmay be a potential therapeutic target for AD treatment. Our future stu-dies using inducible C3 conditional KO mice will determine whetherdepletingC3 after brain development or after the onset ofADpathologywill protect synapses and spare cognitive decline.

MATERIALS AND METHODSStudy designThe objective of our study was to determine whether complement C3plays a role in late-stage AD pathogenesis and cognitive decline. Wecrossbred an AD-like mouse model with C3 KO mice and aged themto 16 months. The APP/PS1;C3 KO mice were compared to APP/PS1,WT, and C3 KO mice in terms of behavioral, neuropathological, andbiochemical end points. Operators were blinded to mouse genotypefor all outcomemeasures.We used power analysis for the water T-mazetest, the outcome measure with the highest variability, to compare cog-nitive behavior between 16-month-oldAPP/PS1;C3KOmice andAPP/PS1 littermates and determined that we needed a minimum of 10 miceper group to achieve statistical significance of P < 0.05 (mean % correctAPP/PS1;C3KO=90.0;mean%correctAPP/PS1= 45.5; SD=10.0;a =0.05; power = 0.80; www.statisticalsolutions.net). To improve the rigor

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of the study, we includedWT (n = 13), APP/PS1 (n = 10 to 11), APP/PS1;C3 KO (n = 11 to 12), and C3 KO (n = 10 to 11) for behavioraltests and n = 6 to 9 per group for biochemical and immunohis-tochemical tests. We excluded the data from one APP/PS1 mousefrom the elevated plus maze test because it was an outlier of approxi-mated 3 SDs from the mean.

MiceC57BL/6J mice, APPswe/PS1dE9 mice, and homozygous C3–deficientbreeder mice (C3−/−; line B6.129S4-C3tm1Crr/J) (63) were obtainedfrom The Jackson Laboratory. C3 KO mice were crossed with APP/PS1 mice to generate APP/PS1 (heterozygotes);C3 KO (heterozygotes)mice, which were backcrossed with C3 KO mice to generate APP/PS1(heterozygotes);C3KO (homozygotes). Thesemice were then bredwithC3 KO mice to generate APP/PS1;C3 KO mice and littermate C3 KOmice. However, a separate cohort of APP/PS1 mice was bred withC57BL/6J mice to generate WT littermates and heterozygote APP/PS1 mice. Mice were genotyped by polymerase chain reaction usingthe following primers: APP/PS1, 5′-GACTGACCACTCGACCAGCTT-3′ and 5′-CTTGTAAGTTGGATTCTCATAT-3′; C3 WT, 5′-ATCTT-GAGTGCACCAAGCC-3′ and 5′-GGTTGCAGCAGTCTATGAAGG-3′;C3mutant, 5′-CTTGGGTGGAGAGGCTATTC-3′ and 5′-AGGTGA-GATGACAGGAGATC-3′.Micewere aged to P30 or 4 or 16months ofage. Only males were used for this study to reduce gender-specific var-iability in the behavioral tests. At the end of the study, mice were an-esthetized, blood was collected, and the brain was perfused with salinebefore harvest.

All animal protocols were approved by the Harvard Medical AreaStanding Committee on Animals, and studies were performed in ac-cordance with all state and federal regulations. The Harvard MedicalSchool animal management program is accredited by the Associationfor the Assessment andAccreditation of Laboratory Animal Care Inter-national and meets all National Institutes of Health standards as de-monstrated by an approved Assurance of Compliance (A3431-01)filed at the Office of Laboratory Animal Welfare.

Water T-mazeThe water T-maze behavioral paradigm assesses spatial learning andmemory by training mice to use the spatial cues in a room to navigateto a hidden platform to escape water. Testing was performed as previ-ously reported (5). Note that the 16-month-old WT and C3 KO miceused for behavioral testing in this large study were the same mice thatwe reported in (5), although they were tested concurrently with theAPP/PS1 and APP/PS1;C3 KO mice in the present study. Here, allanalyses were performed on these same mice.

ImmunohistochemistryHemibrains were fixed in 4% paraformaldehyde for 24 hours, and im-munohistochemistry was performed as described previously (5). Fixed,frozen sections were incubated with anti–Abx–42 (1:200, Covance), 3D6(Ab1–5) (1:1000, Elan), 6E10 (1:1000, Covance), MAP-2 (1:500, Milli-pore), and NeuN (1:200, Serotec) mouse monoclonal antibodies; Ab40(1:200, Covance), Iba-1 (1:200,Wako), andGFAP (1:1000,Dako) rabbitpolyclonal antibodies; or CD68 (1:250, Serotec) rat polyclonal antibodyovernight at 4°C. After washing with tris-buffered saline, sections wereincubated with biotinylated secondary antibodies and developed usingVector ELITE ABC kits (Vector Laboratories) and 3,3′-diaminobenzidine(Sigma-Aldrich) or immunofluorescence-labeled secondary anti-bodies and coverslippedwithmountingmedium (Vector Laboratories).


Thioflavin S staining of amyloid fibrils was performed as previouslydescribed (21).

Ab load analysisImages of Ab immunoreactivity were captured in a single session un-der a Nikon Eclipse E400 microscope. Quantification of Ab plaqueload and analysis of Ab plaque size were measured within an ROIusing the Bioquant image analysis system.

Three of six equidistant planes of 10-mm-thick sections from eachmouse were analyzed. The operator was blinded to mouse genotypeand age when analyzing.

Ab and cytokine ELISAsMSD Ab Triplex ELISA was performed on T-per–insoluble, guanidinehydrochloride–extracted brain homogenates from mouse hemibrain,including cortex and hippocampus. The V-Plex Proinflammatory Cy-tokine ELISA kit (Meso Scale Discovery, catalog no. K15048) was usedfor detecting cytokine levels by MSD plate reader QuickPlex SQ 120(Meso Scale Discovery). Brain homogenization and ELISAs were per-formed as previously described (5).

Confocal analysis of glia morphology and association withAb plaquesImmunofluorescence for Ab plaques (6E10), microglia/macrophages(Iba-1), and astrocytes (GFAP) was detected by confocal microscopy(LSM 710, Carl Zeiss) using a methodology described previously (64).Images were collected using the same exposure settings. Confocal Z-stack images (optical slices of 0.2 mm) of plaques and surrounding gliawere collected using a 63× objective. Three images were acquired fromthree equidistant planes, 500 mm apart, per mouse. Immuno-fluorescence intensity analysis and 3D reconstruction of Z-stack imageswere performedwith confocal image analysis software (ZENBlack, CarlZeiss). Glia counts were performed using stereological methods aftercollecting images.

Synaptic puncta staining and analysisSynaptic puncta staining and analysis were performed as previously de-scribed (5, 65). Confocal imaging was performed using a Zeiss LSM710confocal microscope and a 63× oil objective. Images were acquiredusing a 1 airy unit (AU) pinhole while holding constant the gain andoffset parameters for all sections and mice per experiment.

Preparation of synaptosome fractionsSynaptosome fractions were prepared as described previously (66).

Western blotting of synapse markers and BDNFsignaling proteinsBrain hippocampal and cortical tissues were homogenized, andWesternblotting was performed as previously described (5) using rabbit polyclo-nal antibodies [SYN-1 (Millipore, 1:200), BDNF (Abcam, 1:200), andpCREB and CREB (Chemicon, 1:1000)] and mouse monoclonal anti-bodies [PSD95 (1:200; Millipore) and glyceraldehyde-3-phosphate de-hydrogenase (1:200; Millipore)]. Blots were scanned using the LI-COROdyssey Infrared Imaging System. Intensity of bands was measured byLI-COR Odyssey software.

Stereological quantification of neuron and glia countsImmunohistochemistry for NeuN, Iba-1, and GFAP was performed asdescribed previously (21). Stereological counting of neurons and glia

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was performed on 10-mm sagittal, immunostained sections at each ofsix planes, 250 mm apart, per mouse using the optical dissector meth-od. Cells were counted in an area of ~300 mm within a 10-mm depthper brain region examined. The number of neurons and glia per sec-tion in each brain region was estimated using ~15 optical dissectorsand the Bioquant image analysis system according to the principlesof Cavalieri as described by West and Gundersen (67).

Statistical analysisData are means ± SEM. Comparisons were made betweenWT and C3KO,WTandAPP/PS1, APP/PS1 andAPP/PS1;C3KO, andC3KOandAPP/PS1;C3 KOmice. Significance for all behavioral tests was assessedby one-way ANOVA followed by Fisher’s protected least significantdifference using StatView version 5.0 software. All other data wereanalyzed by one- or two-way ANOVA followed by Bonferroni’s posthoc test or Student’s t test using Prism version 6.0 (GraphPad Soft-ware). P < 0.05 was considered significant.

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SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/392/eaaf6295/DC1Materials and MethodsFig. S1. Behavioral testing of locomotion and anxiety.Fig. S2. C3 deficiency had no effect on Ab deposition in young APP/PS1 mice at the earlieststage of plaque deposition.Fig. S3. Immunoreactivity of Abx–42, Abx–40, and Ab1–x in aged mice.Fig. S4. Glial immunoreactivity was similar in young WT, APP/PS1, and APP/PS1;C3 KO mice atthe earliest stage of plaque deposition.Fig. S5. C3 deficiency resulted in morphological differences in Iba-1–immunoreactive cells inAPP/PS1 aged mice.Fig. S6. C3 deficiency resulted in reduced activation of microglia within Ab plaques in agedAPP/PS1 mice but not in aged J20 mice.Fig. S7. MAP-2–positive dendrites were better preserved within Ab plaques in 16-month-oldC3-deficient APP/PS1 mice versus APP/PS1 mice.Fig. S8. The amounts of APP and PS1 were not significantly different between APP/PS1 andAPP/PS1;C3 KO mice.References (68, 69)

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Acknowledgments: We thank J. Frost, J. Kenison, and Y. Hu [Ann Romney Center for NeurologicDiseases (ARCND)] as well as K. Merry [Boston Children’s Hospital (BCH)] for technical assistance.We thank C. White (ARCND) for statistics advice and B. Liu (ARCND) for data discussions. We aregrateful to S. Matousek (ARCND) and K. Colodner (BCH) for assistance with mouse breeding. WethankD. Selkoe (ARCND) for providing the 3D6antibody for immunohistochemistry andO. Butovsky(ARCND) for critical reading of themanuscript. Funding: This work was funded by Fidelity BiosciencesResearch Initiative (F-PRIME) (C.A.L. and B.S.), NIH/National Institute on Aging (R21 AG044713 toC.A.L.), BrightFocus Foundation Fellowship (Q.S.), and Edward R. and Anne G. Lefler Fellowship (S.H.).Author contributions: C.A.L. and Q.S. co-designed the project and oversaw all experimentation anddata analysis. Q.S. and B.J.C. performed and analyzed the behavior studies. Q.S., S.C., and R.M.performed immunohistochemistry and image analysis for quantification, stereological counts, andELISAs. K.X.L. performed cryosectioning and immunohistochemistry. Q.S. performed confocalimaging and Western blotting. B.S. and S.H. contributed to the discussion of the data. Q.S. andC.A.L. wrote the manuscript with assistance from all authors. Competing interests: C.A.L. serveson the scientific advisory boards of Probiodrug AG, Amgen, and Cognition Therapeutics. B.S.serves on the scientific advisory board and is a minor shareholder of Annexon LLC. The followingpatents related to this project have been granted or applied for: PCT/2015/010288 (S.H. and B.S.):Biomarkers for dementia and dementia-related neurological disorders; US14/988387 andEP14822330 (S.H. and B.S.): Methods of treatment for Alzheimer’s disease and Huntington’sdisease; US8148330, US9149444, US20150368324, US20150368325, US20150368326, andUS20120328601 (B.S.): Modulation of synaptic maintenance. The other authors declare that theyhave no competing interests.

Submitted 5 March 2016Resubmitted 1 October 2016Accepted 15 February 2017Published 31 May 201710.1126/scitranslmed.aaf6295

Citation: Q. Shi, S. Chowdhury, R. Ma, K. X. Le, S. Hong, B. J. Caldarone, B. Stevens, C. A. Lemere,Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1mice. Sci. Transl. Med. 9, eaaf6295 (2017).

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

A. LemereQiaoqiao Shi, Saba Chowdhury, Rong Ma, Kevin X. Le, Soyon Hong, Barbara J. Caldarone, Beth Stevens and Cynthia

DOI: 10.1126/scitranslmed.aaf6295, eaaf6295.9Sci Transl Med

Alzheimer's disease. deposition. Thus, modulation of complement signaling may have potential as a new therapeutic strategy forβ

plaques, possibly by altering the glial response to Aβsynapse loss and cognitive decline even in the presence of Anow report that an aged transgenic mouse model of Alzheimer's disease that lacks C3 was protected against

et al.Alzheimer's disease and, therefore, may contribute to the synapse loss that underlies cognitive decline. Shi the developing visual system by removing weak nerve connections (that is, synapses). C3 is up-regulated in

Complement C3 is an immune molecule that protects against pathogens and plays a role in refinement ofAvoiding complements

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