Rocaglates as dual-targeting agents for experimentalcerebral malariaDavid Langlaisa,b,1, Regina Cencica,b, Neda Moradina,b, James M. Kennedya,b, Kodjo Ayic, Lauren E. Brownd,Ian Crandalle, Michael J. Tarrya, Martin Schmeinga, Kevin C. Kainc, John A. Porco Jr.d, Jerry Pelletiera,b,and Philippe Grosa,b,1

aDepartment of Biochemistry, McGill University, Montreal, QC, H3G 0B1 Canada; bMcGill University Research Center on Complex Traits, McGill University,Montreal, QC, H3G 0B1 Canada; cDepartment of Medicine, Toronto General Hospital-University Health Network, University of Toronto, Toronto, ON, M5G2C4 Canada; dDepartment of Chemistry, Center for Molecular Discovery, Boston University, Boston, MA 02215; and eLeslie Dan Faculty of Pharmacy,University of Toronto, Toronto, ON, M5S 3M2 Canada

Edited by David A. Fidock, Columbia University, NewYork, NY, and accepted by Editorial BoardMember Carl F. Nathan January 19, 2018 (received for review July 24, 2017)

Cerebral malaria (CM) is a severe and rapidly progressing complica-tion of infection by Plasmodium parasites that is associated with highrates of mortality and morbidity. Treatment options are currentlyfew, and intervention with artemisinin (Art) has limited efficacy, aproblem that is compounded by the emergence of resistance to Art inPlasmodium parasites. Rocaglates are a class of natural productsderived from plants of the Aglaia genus that have been shown tointerfere with eukaryotic initiation factor 4A (eIF4A), ultimately block-ing initiation of protein synthesis. Here, we show that the rocaglateCR-1-31B perturbs association of Plasmodium falciparum eIF4A(PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and thera-peutic antiplasmodial activity in vivo in mouse models of infectionwith Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical iso-lates of P. falciparum in human erythrocytes infected ex vivo, includ-ing drug-resistant P. falciparum isolates. In vivo, a single dosing ofCR-1-31B in P. berghei-infected animals is sufficient to provide pro-tection against lethality. CR-1-31B is shown to dampen expression ofthe early proinflammatory response in myeloid cells in vitro and damp-ens the inflammatory response in vivo in P. berghei-infected mice. Thedual activity of CR-1-31B as an antiplasmodial and as an inhibitor of theinflammatory response in myeloid cells should prove extremely valu-able for therapeutic intervention in human cases of CM.

Plasmodium | cerebral malaria | severe malaria | protein translation |dual target

Malaria is a severe threat to global health, with an estimated200–300 million cases annually and 445,000 deaths in 2016;

it accounts for ∼25% of pediatric deaths in endemic areas ofAfrica (1–3). Malaria is also a significant threat to immunologicallynaive travelers visiting or working in these areas (4). It is caused byinfection with members of the Plasmodium parasite family, withclinical symptoms occurring at the blood stage, when Plasmodiummerozoites rapidly replicate and lyse erythrocytes (RBCs), leading toanemia and high fever (2, 3). Furthermore, parasitized RBCs(pRBCs) can adhere to the brain microvasculature, causing localinflammatory and vascular tissue damage, which, in turn, leads tocerebral malaria (CM), a severe syndrome that can rapidly evolve tofatal encephalomyopathy and is mainly caused by Plasmodium fal-ciparum (5, 6). Despite antimalarial treatment, CM has a high fatalityrate (18–40%) and up to one-third of survivors are afflicted withneurocognitive deficits (7). The malaria problem is further compli-cated by widespread parasite resistance to antimalarial drugs (8–12),resistance of the Anopheles vector to insecticides (13), and the ab-sence of an effective antimalaria vaccine despite enormous researchefforts (14).Treatment of clinical malaria (uncomplicated disease) relies

entirely on antimalarial drugs, with artemisinin (Art) being themost effective drug currently available (8). To avoid emergence ofArt resistance, Art monotherapy is discouraged and has beenreplaced by Art combination therapy (ACT). ACTs are the cur-

rent WHO-approved standard of care for uncomplicated malaria.ACTs include Art (artesunate, dihydro-Art, artemether, andarteether) in combination with other drugs with pharmacokineticprofiles showing longer circulation half-lives, such as mefloquine,lumefantrine, amodiaquine, piperaquine, and sulfadoxine/pyri-methamine (15, 16). In contrast, treatment options for CM arefew and are effective only when infection is detected early beforeirreversible neurological symptoms (5–7). Administration of i.v.artesunate is the WHO-approved first-line therapy, but efficacyis limited, with 18–30% of CM cases still being fatal in largerandomized clinical trials in children and adults (17–19). Becauseof the lack of equipotent drug alternatives, the recent emergenceand spread of Art resistance in different areas of Southeast Asia is amajor threat to global health (8, 12). Although the mechanisms ofArt resistance in Plasmodium are being characterized (20–24), it isunclear that novel treatment options based on this knowledge willbe immediately forthcoming. Thus, there is a great urgency to de-velop new therapeutic approaches to overcome Art resistance in themalarial parasite. Likewise, increasing effectiveness of Art-basedtherapies may improve the clinical outcome of CM, the most se-vere and most difficult-to-treat complication of malaria.Recent studies have pointed to initiation of protein synthesis as

a novel pharmacological target in P. falciparum for the develop-ment of effective antimalarial drugs (25–27). Considering the

Significance

Severe complications of malaria, such as anemia and cerebralmalaria (CM) (neuroinflammation), are responsible for ∼25% ofinfant mortality in certain regions of Africa. Notwithstandingcurrent effective antiplasmodial treatment, drug resistance is onthe increase in the Plasmodium parasites, and therapy to treatCM is nonexistent. Here, we show that rocaglates derived from anatural product originally identified from plants of the Aglaiaspecies, effectively block blood-stage parasite replication inseveral mouse models and in infected human RBCs. Importantly,neurological inflammation is mitigated and survival is promotedin experimental CM, highlighting the strong potential of thisclass of compounds to treat complicated malaria.

Author contributions: D.L., J.P., and P.G. designed research; D.L., R.C., N.M., J.M.K., K.A.,L.E.B., I.C., M.J.T., M.S., K.C.K., and J.A.P. performed research; D.L., M.S., and J.A.P. con-tributed new reagents/analytic tools; D.L., R.C., N.M., J.M.K., K.A., K.C.K., J.P., and P.G.analyzed data; and D.L., K.C.K., J.A.P., J.P., and P.G. wrote the paper.

Conflict of interest statement: A provisional patent application based on the technologydescribed in this work has been filed.

This article is a PNAS Direct Submission. D.A.F. is a guest editor invited by the Editorial Board.

Published under the PNAS license.1To whom correspondence may be addressed. Email: david.langlais@mcgill.ca or philippe.gros@mcgill.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1713000115/-/DCSupplemental.

Published online February 20, 2018.

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functional and structural conservation of components of thetranslation initiation machinery, we reasoned that known mam-malian translation initiation inhibitors may display activity againsttheir Plasmodium counterparts, including antimalarial activity invivo. One target of this pathway that has been extensively scruti-nized for small-molecule discovery is eukaryotic initiation factor4F (eIF4F), a heterotrimeric complex required for ribosome re-cruitment to capped (m7GpppX; X is any nucleotide) mRNAs(28). eIF4F is composed of eIF4E, the cap-binding subunit;eIF4A, a DEAD-box RNA helicase required to remodel themRNA 5′ leader region; and eIF4G, responsible for recruitingthe small ribosomal subunit (and associated factors) to themRNA (28). Rocaglates, the most characterized and potentsmall molecules found to target eIF4F (29), are a class of naturalproducts derived from plants of the Aglaia genus (30) that in-terfere with eIF4A activity in two ways. Rocaglates cause eIF4Ato clamp onto RNA, which, if it occurs in the mRNA 5′ leaderregion, may act as a steric barrier to the initiation process, andthey deplete the eIF4F complex of its eIF4A helicase subunit(31–34). The net result is reduced initiation complex formation.Since eIF4F discriminates between mRNAs during the initiationprocess, rocaglates, unlike general inhibitors of elongation, exerta selective inhibitory effect on a subset of translating mRNAs(31–34). They display tolerable safety profiles in vivo and exhibitanticancer properties in tumor xenograft models (31, 35). Inaddition, certain rocaglates can interfere with nuclear factor ofactivated T cells (NFAT) and nuclear factor-kappa B (NF-κB)signaling pathways in T cells in vitro (36, 37), suggesting po-tential antiinflammatory properties.In the present study, we have investigated the possible anti-

plasmodial activity of these compounds in vivo in mouse models ofblood-stage malaria (Plasmodium chabaudi) and experimental ce-rebral malaria [ECM; Plasmodium berghei ANKA (PbA)], as wellas against multiple drug-resistant P. falciparum lines in vitro. Weshow that rocaglates are potent antimalarial drugs, demonstratingboth direct antiplasmodial and antiinflammatory activities in vivo.Single dosing is sufficient to protect mice against ECM induced byPbA. Our data suggests that this effect is a consequence of roca-glates suppressing both the activity of Plasmodium falciparumeIF4A (PfeIF4A) and the excessive host inflammatory response.

ResultsRocaglates Interact with PfeIF4A. We investigated translation ini-tiation as a potential new target for antimalarial drug discovery.We noted a very strong sequence conservation (70% identity,83% total similarity) between mammalian eIF4A1 and PfeIF4Athat extended beyond the conserved signature motifs that defineDEAD-box helicase family members (Fig. S1). Of note, humanand mouse parasites have near-complete sequence identityconservation (Fig. S1).We asked whether this strong structural similarity translated to

functional conservation, including interaction with small-moleculemodulators of mammalian eIF4A. Members of the rocaglatesfamily, characterized by a common cyclopenta[b]benzofuran skel-eton (Fig. 1A), have been shown to be among the most potentinhibitors of eIF4A, and structure–activity relationships haveidentified highly potent translation inhibitors from within this family(33, 35). CR-1-31B and (−)-SDS-1-021 act by increasing clampingof eIF4A onto RNA with a concomitant decrease in eIF4F-associated eIF4A, ultimately leading to impaired 43S ribosomeloading onto mRNA (31–35) (Fig. 1B). For both molecules, onlythe (−) enantiomers are active; the molecules used in the currentstudy were prepared as single, bioactive enantiomers using ourpreviously published methods (38, 39) [Fig. S2; >95% pure com-pounds, as confirmed by ultraperformance liquid chromatography(UPLC)/evaporative light scattering detection (ELSD) analysis].We tested whether PfeIF4A could also be targeted by roca-

glates. Both murine eIF4A1 (meIF4A1) and PfeIF4A were

expressed as recombinant proteins and purified from Escherichiacoli (Fig. 1C), and their ability to interact with [32P]-labeled RNAwas quantified in the presence and absence of two rocaglates:silvestrol and the hydroxamate CR-1-31B (Fig. 1 D and E). BotheIF4A proteins showed similar behavior, including low-levelRNA binding under control conditions, which was significantlystimulated by silvestrol and CR-1-31B (approximately six- to 10-fold increase). Clamping of eIF4A onto RNA, especially when inthe context of 5′ leaders, has the potential to block ribosomescanning and could be one contributor to the inhibition oftranslation imparted by these compounds. We directly tested theeffect of CR-1-31B on parasite protein synthesis by metaboliclabeling using [35S]-methionine incorporation in PbA pRBCs(Fig. 1 F and G). In these experiments, cycloheximide (CHX)was used as a positive control, while the antimalarial drugchloroquine (CQ), which does not target translation, was used asa negative control (Fig. 1 F and G). The effect of CR-1-31B onprotein synthesis was monitored directedly by SDS/PAGE ofP. berghei-labeled cell extracts and quantified by scintillationcounting. CR-1-31B could inhibit P. berghei protein synthesispotently with an IC50 in the low nanomolar range, proving to be abetter inhibitor than CHX (Fig. 1 F and G). In addition, we havemeasured the level of P. falciparum lactate dehydrogenase(pLDH; the terminal enzyme of their glycolytic pathway) pro-duced in in vitro cultures exposed to increasing concentrations ofCR-1-31B or CHX. The level of pLDH is potently reduced byCR-1-31B with an IC50 of ∼3 nM, compared with an ∼300 nMIC50 for CHX in both 3D7 and ItG P. falciparum isolates (Fig.S3). Altogether, these initial observations strongly suggest thatrocaglates can efficiently interfere with PfeIF4A activity andassociated protein synthesis in a manner similar to their dem-onstrated action on mammalian eIF4A.

Antiplasmodial Activity of Rocaglates in a Mouse Model of CM. Thepotential antiplasmodial activity of rocaglates was assessed invivo in a murine model of ECM induced by infection with PbA.In this model, mice infected with PbA develop blood-stage para-sitemia detectable within 2 d. PbA-parasitized erythrocytes becometrapped in the brain microvasculature, leading to a pathologicalinflammatory response in situ and appearance of drastic cerebralsymptoms (paralysis, seizure, and coma), with a clinical end pointreached by day 5–6 postinfection. Cerebral disease typically de-velops with levels of blood parasitemia <10%. Using a prophylactictreatment regimen where mice received daily dosing for 10 d(starting at day −2 and ending at day +8), we observed thattreatment with the structurally related rocaglates CR-1-31B(0.2 mg/kg) and SDS-1-021 (0.05 mg/kg) (Fig. 2A) almostcompletely protected mice from ECM, with 80–90% oftreated mice surviving beyond day 15, while untreated micesuccumbed by day 6. Blood parasitemia monitored at days3 and 6 postinfection showed a significant reduction of Plas-modium-infected RBCs (pRBCs) in both the CR-1-31B– andSDS-1-021–treated groups (Fig. 2B). These data indicate thatprophylactic treatment of mice with rocaglates can preventand significantly reduce mortality in the ECM model.We next tested the capacity of rocaglates to act therapeutically

and cure an established PbA infection. For this, treatment ofPbA-infected mice with CR-1-31B (0.2 mg/kg) was performeddaily starting at various times postinfection (day 1, day 3, and day4) and continuing to day 8 with disease outcome monitored. CR-1-31B could significantly improve survival from PbA, even whentreatment was initiated later in the course of infection up to day+4 (Fig. 2C). Similar results were obtained using SDS-1-021 (Fig.S4). With both SDS-1-021 and CR-1-31B, the protective effect ofrocaglates was correlated with length of treatment and total dosereceived (Fig. 2C and Fig. S4) and was associated with a signif-icant reduction in blood parasitemia (measured at days 3, 6, and8 postinfection; Fig. 2D). Interestingly, in CR-1-31B–treated

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animals, parasitemia measured at day 8 was lower than at day 6,indicating that continued treatment with CR-1-31B caused areduction of parasitemia from the peak measured at day 6 (Fig.2D). Finally, the therapeutic effect of CR-1-31B was found to bedose-dependent: Animals dosed daily (starting at day 3 andcontinuing to day 8) with CR-1-31B at either 0.1 or 0.2 mg/kgwere protected against lethal PbA-induced CM, while animalsreceiving a lower daily dose of 0.02 mg/kg were as susceptible asuntreated controls (Fig. 2E). Parasitemia levels were also af-fected in a dose-dependent manner (Fig. 2F and Fig. S5), withthe lower 0.02-mg/kg dose having no effect, while 0.1- and0.2-mg/kg dosing caused a dose-dependent decrease in parasite

growth (measured at day 5 postinfection). These data suggestthat therapeutic intervention with rocaglates can prevent andsignificantly reduce mortality in the ECM model.

Antiplasmodial Activity of Rocaglates Against Blood-Stage Parasites.We conducted additional experiments to further validate theeffect of the rocaglate CR-1-31B on blood-stage replication ofthe malarial parasite. First, we monitored the course of bloodparasitemia in a mouse mutant that does not develop CM fol-lowing PbA infection due to an inactivating mutation in theThemis gene, which causes impairment of T lymphocyte-drivenneuroinflammation (40). Control untreated Themis mice infected

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Fig. 1. Rocaglates stabilize the interaction between PfeIF4A and mRNA. (A) Chemical structure of rocaglates used in this study: silvestrol, CR-1-31B, and(−)-SDS-1-021. (B) Mechanism by which rocaglates inhibit translation initiation (stabilization of the eIF4A/RNA complex leads to depletion of the eIF4Ahelicase from the eIF4F complex and eIF4A clamped onto mRNA, which may interfere with ribosome scanning); components of relevant proteins of thetranslation initiation machinery are identified. AUG, translation initiation codon. (C) Analysis of murine eIF4A1 (meIF4A1) and PfeIF4A recombinant proteinsby gel electrophoresis and Coomassie blue staining. (D and E) Effect of silvestrol and CR-1-31B on binding and retention of purified meIF4A1 and PfeIF4Arecombinant proteins to radiolabeled RNA assessed by filter-binding assay; significance was determined using an unpaired two-tailed Student’s t test for theindicated pairs (*P < 0.05; **P < 0.01). (F and G) Incorporation of [35S]-methionine to assess protein synthesis in PbA-infected blood and to test the effect ofincreasing doses of CR-1-31B, CHX, or CQ. (F) One sample per treatment condition was resolved on SDS/PAGE, and radiolabeled proteins were revealed byautoradiography; noninfected blood was included as a labeling control. (G) Experiment was performed twice in triplicate, and pooled data are presented asmean ± SD of the [35S]-methionine incorporation as counts relative to vehicle-treated parasites.

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with PbA do not develop cerebral symptoms and survive past day 6(compared with B6 controls) (Fig. 3A), but they do sustain pro-gressive blood-stage parasite replication, leading to very high levelsof blood parasitemia by day 15 (>40%). Treatment of Themismicewith CR-1-31B (0.2 mg/kg) starting at either day 3 or day 5 post-infection and continuing to day 11 caused a significant reduction inblood parasitemia compared with untreated mice (Fig. 3B). Sec-ond, we investigated the effect of CR-1-31B on replication of P.chabaudi AS (PcA). PcA is a blood-stage parasite that does notinduce CM (41), with infected mice rather succumbing to hyper-parasitemia and associated severe anemia by day 10 (Fig. 3 C andD). Treatment of PcA-infected mice with CR-1-31B (0.2 mg/kg,standard 4-d test) fully protected animals against lethality fromPcA infection. CR-1-31B–induced protection was associated witha highly significant reduction in blood parasitemia measured dailybetween days 3 and 6 postinfection (Fig. 3D), an effect that wascomparable to protection afforded by treatment with high-doseartesunate (1.5 mg/kg) (Fig. 3D). These results strongly supporta direct antiplasmodial effect for CR-1-31B in vivo against mu-rine Plasmodium parasites that induce blood-stage infection orcerebral disease.

Antiplasmodial Activity of Rocaglates Against Human P. falciparumParasites. In dose–response studies with human erythrocytes in-fected in vitro, we also monitored CR-1-31B activity against thehuman P. falciparum malarial parasite (Fig. 4 A and B), usingpreviously described experimental conditions (42). In these ex-periments, we used a number of different P. falciparum isolates,including four freshly isolated from patients seen at the Uni-versity of Toronto Health Network (UHN1–4), standard labo-ratory lines (3D7, ItG, CS2, and FCR3), and isolates fromThailand that display low levels of CQ and mefloquine resistance(MS-97 lines). Infected blood cultures were exposed to variousconcentrations of CR-1-31B or CQ for 48 h, and the effect ofdrug exposure on parasite growth inhibition was monitored andquantified (IC50) using a fluorescence-based assay (SYBR-GreenI) (Fig. 4 A and B). CR-1-31B showed strong activity against allP. falciparum lines tested, with IC50 values in the low nanomolarrange, varying between 0.9 and 2.8 nM, whereas CQ had an IC50

average of ∼100 nM (Fig. 4C). Together, these results demon-strate that CR-1-31B acts to block replication of human malarialparasites, arguing for a possible therapeutic value of CR-1-31Bin clinical management of malaria.

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Fig. 2. Rocaglates protect mice from CM. Mice infected with PbA were left either untreated or treated with different daily i.p. regimens (triangles) of CR-1-31B or(−)-SDS-1-021, and the extent of parasite replication in blood (percentage of infected erythrocytes measured at the indicated days postinfection),appearance of neurological symptoms (CM phase, shaded area), and overall survival (percentage of surviving mice, total number of mice tested indicated)were monitored. (A and B) Effect of prophylactic treatment regimen with CR-1-31B and (−)-SDS-1-021 on PbA infection parameters (survival and blood-stageparasitemia). (C and D) Effect of different therapeutic treatment regimens (initiated at different days postinfection and given i.p.) with CR-1-31B on PbAinfection parameters. (E and F) Effect of different doses of CR-1-31B (0.02, 0.1, and 0.2 mg/kg) in a therapeutic regimen initiated at day 3 on PbA infectionparameters. Survival data were compared using the Mantel–Cox test, and significance was established relative to the vehicle (Veh)-treated mice; parasitemiavalues were compared by ANOVA, followed by a Bonferroni posttest (indicated level of significance: *P < 0.05; **P < 0.01; ***P < 0.001). Ctrl, control.

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Effect of CR-1-31B on Host Inflammatory Response in Vivo. It is highlydesirable for new antimalarials to be effective once clinicalsymptoms have started and, ideally, to be efficacious at a singledosing. Therefore, we tested whether treatment with CR-1-31Blate during infection could blunt the development of disease andcure infected animals. We observed that treatment of PbA-infected mice with CR-1-31B starting at day 5 (when the firstneurological symptoms appear) and continuing until day 8 causedsignificant protection against ECM in these mice (Fig. 5A). Fur-thermore, we observed that even a single dosing of CR-1-31B ateither 0.2 or 0.5 mg/kg administered on day 5 had beneficial ef-fects on disease and, at the higher dose, caused a significant in-crease in overall survival of infected mice (Fig. 5B). Thisintervention is similar to what is needed in human cases of CM,where high mortality is associated with rapidly evolving and irre-versible neurological symptoms. Finally, we note that the observedtherapeutic effect of a single dose of CR-1-31B given at day5 occurs in the absence of a strong reduction in parasite load andassociated blood parasitemia measured at day 5 or 6 (Fig. 5C).Excessive inflammatory response in situ is a major pathological

feature of ECM. This response is mediated, in part, by infiltrationof activated leukocytes in the brain, with concomitant productionof proinflammatory cytokines. Cells of the myeloid lineage play acritical role in this pathological response, and mouse mutantsharboring reduced numbers or impaired activity of these cellsconfer resistance in the ECMmodel (43, 44). The observation thatvery late treatment with CR-1-31B can protect PbA-infected miceagainst CM without affecting blood parasitemia (Fig. 5C) led usto test the possibility that CR-1-31B may also have an impact onthe host inflammatory response in situ in the brain. Neuro-inflammation in ECM is associated with breakdown of the blood–brain barrier (BBB). An Evans Blue dye extravasation assay wasused to assess BBB integrity in PbA-infected mice (Fig. 5 D andE). As opposed to the brains of PbA-infected B6 controls thatcannot exclude the blue dye, brains from CR-1-31B-treated mice

remained largely unstained, a feature that is indicative of an intactBBB and is similar to what is observed in the ECM-resistantmouse mutant, Irf8R294C (43) (Fig. 5 D and E). By flow cytom-etry analysis, we observed reduced cellular infiltration of leuko-cytes (Fig. S6A), of both CD11b+ myeloid cells (Fig. 6 A and B)and TCRb+ lymphoid cells (Fig. 6 D and E and Fig. S6 B and C),in the brain of CR-1-31B–treated mice compared with controlPbA-infected untreated mice. Reduced infiltration of inflamma-tory cells in the brain coincided with reduced RNA expression ofgenes encoding for the myeloid (Cebpd and C4b) and for thelymphoid cell lineages (Gzmb, Cd3g, and Ifng) (Fig. 6 C and F andFig. S6D). Hence, ECM resistance in mice treated with CR-1-31Bcorrelates with reduced infiltration of leukocytes in the brain, re-duced expression of proinflammatory genes in situ, and preser-vation of BBB integrity.

Effect of CR-1-31B on Host Inflammatory Response in Vitro. Finally,we directly tested the effect of different doses of CR-1-31B onactivation of isolated primary myeloid cells in response to IFN-γ,which was monitored both by induction of the master proin-flammatory transcription factor IRF1 and by production of earlyinflammatory markers (GBP2 and CXCL10) (Fig. 7). The proteinlevel of STAT1, another proinflammatory transcription factordownstream of IFN signaling that forms transcription complexeswith members of the IRF and ETS families to activate transcrip-tion in response to proinflammatory stimuli (45), was not affectedby CR-1-31B exposure (Fig. 7A). On the other hand, exposure ofbone marrow-derived macrophages (BMDMs) to CR-1-31B sup-pressed production of IRF1 (Fig. 7), a major transcriptional acti-vator mediating myeloid cell activation in response to type I andtype II interferons, as well as other viral and bacterial stimuli. Thiswas associated with reduced expression of protein markers of ac-tivation in these cells (GBP2) and production of the critical earlyinflammatory chemokine CXCL10 (Fig. 7). The effect of CR-1-31B on inflammatory protein production was dose-dependent and

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Fig. 3. Rocaglate CR-1-31B blocks blood-stage replication of different Plasmodium malarial parasites. (A and B) Immunodeficient ThemisI23N mutant mice(Themis) do not develop CM symptoms following PbA infection (as shown in A), and were used to assess the effect of different therapeutic i.p. regimens ofCR-1-31B (initiated at day 3 and day 5; green and red triangles, respectively) on blood-stage parasite replication. (C and D) Effect of CR-1-31B (0.2 mg/kg i.p.)on parameters of infection with the blood-stage parasite PcA were determined and compared with that of Art (1.5 mg/kg i.p.) using a standard 4-d test (57)and PcA-suceptible A/J mice. Statistical analyses were done as described in the legend for Fig. 2 and compared drug-treated groups with groups receivingvehicle (Veh) only (*P < 0.05; ***P < 0.001).

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was not due to transcriptional inhibition. These results suggest thatrocaglates can also dampen host inflammatory function in myeloidcells, with a beneficial impact on neuroinflammation that drivespathogenesis in the ECM model.

DiscussionRocaglates (flavaglines) are a class of natural products that wehave previously characterized as novel anticancer drugs (29).These compounds act as potent inhibitors of translation initia-tion by targeting eIF4A in two ways. They cause clamping of freeeIF4A onto RNA, resulting in complexes that may be sufficiently

stable to impair ribosome scanning if they occur in the mRNA 5′leader regions and if they deplete eIF4A from the eIF4F com-plex, resulting in decreased initiation events (31–34). Impor-tantly, systemic suppression of eIF4F is well-tolerated at theorganismal level, increasing the therapeutic potential of roca-glates (31, 46, 47). Recent studies have suggested that translationis a potential target for antimalarial drug discovery (26). Indeed,2,6-disubstituted quinoline-4-carboxamide analogs show strongactivity against different life forms of the human P. falciparumparasite, targeting translation elongation factor 2 (PfEF2) (26).Considering the high degree of structural and functional con-servation among proteins involved in the translation machinery,we investigated the possibility that rocaglates may (i) interactwith PfeIF4A and (ii) display antimalarial activity in in vivo andex vivo models of infection.We obtained strong experimental evidence indicating that the

rocaglate CR-1-31B has direct antimalarial activity. First, wedemonstrate that CR-1-31B can stabilize complexes formed invitro between recombinant PfeIF4A and RNA, and this in amanner similar to mammalian eIF4A1 tested under the sameconditions. Second, Plasmodium protein synthesis is inhibited bylow nanomolar concentrations of CR-1-31B in a 1-h radioactivemetabolic labeling assay and in a 24-h pLDH assay. Third, CR-1-31B can block replication of P. falciparum in human erythrocytesinfected ex vivo, with IC50 values in the low nanomolar range.Importantly, CR-1-31B shows similar IC50 values for P. falcipa-rum clinical isolates from different regions of the world thatdisplay diverse degrees of resistance to established antimalarialdrugs. Fourth, when tested in prophylactic and therapeuticprotocols, CR-1-31B protected mice against lethal CM caused byinfection with P. berghei. In all cases, the antimalarial effect ofCR-1-31B is dose- and exposure-dependent and is associatedwith a significant decrease in blood parasitemia in treated mice.Fifth, CR-1-31B is also active against P. chabaudi, a strict blood-stage Plasmodium parasite that replicates rapidly in blood butdoes not cause CM. Finally, it is important to note that a singledose of CR-1-31B administered to P. berghei-infected mice at day5, a time point at which control untreated infected mice displayearly neurological symptoms (lethargy), is sufficient to signifi-cantly improve survival and BBB integrity. Both single-dose cureand novel modes of action with no cross-resistance to currentdrugs are two key selection criteria in the global search for novelantimalarial drugs (48, 49). Taken together, these studies es-tablish that CR-1-31B has direct antiplasmodial activity by actingon PfeIF4A, but we cannot formally exclude that CR-1-31B mayalso affect other targets.The rocaglate CR-1-31B was previously shown to have good

pharmacokinetic properties (35) that include high solubility andmembrane permeability at physiological pH. Incubation of CR-1-31B in human plasma showed that it was 82–84% bound toplasma proteins and that it was highly stable (100% remainingafter 3 h). Experiments with murine and human liver microsomesdemonstrated that CR-1-31B has a significant hepatic stability(>95% after 1 h). In mice receiving repeated daily injections at0.2 mg/kg, no observable toxic effects were detected and CR-1-31B did not display toxicity toward cells of hematopoietic origin(35). Altogether, these preliminary CR-1-31B pharmacologicaland safety data suggest possible use in humans for CM.Our studies extend those of Bhattacharya et al. (50) in doc-

umenting an antiinflammatory response by rocaglates. Impor-tantly, we demonstrate that CR-1-31B, and related rocaglates, canbe used to interdict this process in the brain, highlighting its po-tential use as an antimalarial drug for intervention in CM in hu-mans. An antiinflammatory effect of CR-1-31B to dampenneuroinflammation was initially suggested by results from single-dosing experiments, where dramatic improvements in survival ofPbA-infected CR-1-31B–treated mice (i) occurred in the absenceof a substantial reduction in blood parasitemia (Fig. 5), (ii) were

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associated with decreased infiltration of myeloid and lymphoidinflammatory cells in the brain and maintenance of the BBB in-tegrity (Figs. 5 and 6), and (iii) led to reduced cerebral expressionof molecular markers of proinflammatory cells and inflammatorymediators (i.e., Cebpd, Cd3g, Ifng, C4b, Gzmb) at the peak ofneuroinflammation (Fig. 6 and Fig. S6D). Finally, we showed thatexposure of myeloid cells to CR-1-31B causes a significantdampening of the inflammatory response of these cells followingexposure to IFN-γ, as demonstrated by reduced protein expressionof the master transcriptional regulator IRF1 and the inflammatorychemokine CXCL10 by these cells (Fig. 7). These findingsestablish a rapid and beneficial antiinflammatory activity ofCR-1-31B during PbA-induced experimental CM and associatedneuroinflammation.A possible explanation of these unexpected findings is that CR-

1-31B–mediated inhibition of mammalian eIF4A/eIF4F prefer-entially inhibits translation of mRNAs encoding proinflammatoryeffectors. There is a growing body of literature suggesting thatearly activation of proinflammatory pathways involves regulationat the level of translation, including proteins that are themselveskey transcriptional activators of the inflammatory response (51–53). For example, Colina et al. (54) showed that translationalcontrol is critical for expression of early defenses, including type IIFN. They observed increased resistance to a number of viral in-fections in 4E-BP1/2−/− mice, two redundant translational re-pressors of eIF4F, and that this was associated with increasedproduction of type I IFN. They further demonstrated that 4E-BP1/2 inactivation caused increased translation of the type I IFNtranscriptional activator IRF7 from existing RNA pools in plas-macytoid dendritic cells. This establishes that the translationalrepressors 4E-BP1/2 act as negative regulators of type I IFN andthat the translation of IRF7 is regulated by eIF4F (54). It is

tempting to speculate that inhibition of eIF4A by CR-1-31B couldlikewise dampen early proinflammatory responses such as type I IFN,thereby benefiting the host under situations that arise such as CM,where pathogenesis is driven by acute inflammation. Indeed, we andothers have previously reported rapid activation of the IFN responsein situ in the brain of PbA-infected mice, and have shown that in-activation of Irf7 and other genes that are part of this response (IFN-stimulated genes:Usp15, Trim25, Stat1, Irf1, Irf3, Irf8, and Socs1/Socs3)causes resistance to CM (43, 55, 56). These findings are consistentwith the proposal that inhibition of translation initiation in general,and of eIF4A in particular, dampens the inflammatory response.Our studies also tease out an important functional property of

rocaglates in Plasmodium-infected mice: the ability to act as dual-targeting agents displaying strong antimicrobial activity against thisparasite while also dampening the acute inflammatory response ininfected brains of the host. The end result is a potent perturbationof parasite replication and pathogenesis at low nanomolar con-centrations. These properties, documented in preclinical modelsof blood-stage and cerebral disease, make CR-1-31B a high-profilecandidate for intervention in CM in humans. The efficacy of CR-1-31B and other rocaglates against human malaria will requirefuture testing in clinical studies either alone or in combinationwith Art, which is the current standard of care for CM.

Materials and MethodsMice and Murine Plasmodium Parasites. Eight-week-old female C57BL/6 micewere purchased from the Charles River Laboratories. Mouse mutant strainscarrying loss of function at either Irf8 (Irf8R294C; Irf8m/m) (43) or Themis(ThemisI23N) (40) are on a C57BL/6 background and are resistant to CM aspreviously described. All mice were housed at McGill University Animal CareCenter according to the guidelines of the Canadian Council on Animal Care.All protocols were approved by the Animal Care Committee of McGill Uni-versity (Protocol 5287), and include procedures to minimize distress and

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Fig. 5. CR-1-31B–mediated protection against neuroinflammation in late-stage CM. (A) Effect of a therapeutic regimen with CR-1-31B (0.2 mg/kg; four dailyi.p. injections) initiated at day 5 post-PbA infection on survival (significance determined using the Mantel–Cox log-rank test: ***P < 0.001). Veh, vehicle.(B and C) Effect of late-stage, single high doses of CR-1-31B (0.2 mg/kg and 0.5 mg/kg) given i.p. at day 5 on infection parameters in PbA-infected mice. Theeffects on survival (B) and on blood-stage parasitemia (C) are shown. Parasitemia levels were compared using ANOVA with a Bonferroni posttest. (D) Integrityof the BBB in normal mice (NI) and PbA-infected mice treated with Veh or CR-1-31B (single 0.5-mg/kg dose, i.p., day 5) was assessed by Evan’s Blue ex-travasation assay. The CM-resistant Irf8R294C mutant mice (43) were included as positive controls. (E) Evan’s Blue dye accumulation in the brain was quantifiedand expressed as micrograms per gram of brain tissue. Data are presented as mean ± SD, and groups were compared using an unpaired Student’s t test (*P <0.05; **P < 0.01; ***P < 0.001).

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improve welfare. An isolate of PcA was provided by Mary M. Stevenson,McGill University Health Center Research Institute, and was maintained byweekly passage in A/J mice for a maximum of four consecutive passages, aspreviously described (41). An isolate of PbA was initially obtained from theMalaria Reference and Research Reagent Resource Center (MR4); PbA waspassaged in C57BL/6J (B6) mice for a maximum of three consecutive passages(57). In passage mice, blood parasitemia was monitored by calculating thepercentage of pRBCs on thin blood smears stained with Diff-Quick (DadeBehring), as described (41). Infected blood was diluted in pyrogen-free salinefor infection of experimental animals.

Human Plasmodium Parasites. Several different P. falciparum isolates wereused to test the antimalarial activity of CR-1-31B. These included four freshlyisolated clones from patients seen at The University of Toronto Health Networkof the University of Toronto (UHN1–4). We also tested standard laboratory lines3D7, ItG, CS2, and FCR3, as well as recent isolates from Thailand showing a lowlevel of CQ and mefloquine resistance (MS-97 isolates).

All P. falciparum clones were mycoplasma-free and were maintained incontinuous culture with fresh O+ erythrocytes from healthy blood donors aspreviously described (58). Ring-form cultures were synchronized with 5%sorbitol treatment twice over two intraerythrocytic cycles (59). To assess theantimalarial activity of the drugs tested, we used a SYBR-Green method toquantify parasite genomic DNA (60). Briefly, a 96-well plate with a series oftwofold dilutions was produced containing R-10G medium plus drug and R-10G medium alone (last well). The parasite culture, 1% hematocrit and 2%parasitemia, was added to each well, and the plates were incubated at 37 °Cand gassed (93% N2, 5% CO2, and 2% O2) for 48 h. Relative fluorescence wasassessed using a Biotek Synergy-4 Multi-Mode Microplate Reader (BiotekInstruments, Inc.) and a Fluostar Optima plate reader (BMG Labtech). TheIC50 values of CR-31 or CQ for individual isolates were determined using afour-variable nonlinear regression analysis of the relative fluorescence ofindividual wells following the addition of SYBR-Green I using GraphPadPrism v7.0 software.

In vitro P. falciparum protein inhibition was performed by using a Plas-modium-specific LDH assay (61) after a 24-h incubation period and de-termining the absorbance at 595 nm using a plate reader. Briefly, compounds

were dissolved in DMSO solution before being used to prepare serial twofolddilutions across a 96-well plate. Parasite culture material was then added togive a final hematocrit of 2% and a parasitemia of 2%, and the plate was thenincubated for 24 h at 37 °C in an atmosphere of 95% N2, 3% CO2, and 2% O2.The IC50 values of CR-1-31B and CHX were determined using a four-variablenonlinear regression analysis using GraphPad Prism v7.0 and represent themean ± SEM (n = 4).

Protein Sequence Alignment. Human (NP_001407.1) and murine (NP_659207.1)eIF4A1 protein sequences were retrieved from the National Center for Bio-technology Information Refseq database. The murine eIF4A1 protein sequencewas used as input in the PlasmoDB BLASTp tool to identify similar proteins inPlasmodium parasites. Putative PfeIF4A proteins were identified in all humanand murine parasites, having 70% sequence identity and 83–84% sequencesimilarity. Plasmodium protein sequences were downloaded from PlasmoDB (62)for P. falciparum 3D7 (PF3D7_1468700), Plasmodium vivax Sal-1 (PVX_117030),Plasmodium knowlesi strain H (PKNH_1212500), PbA (PBANKA_1331900), andP. chabaudi AS (PCHAS_1336500). Multiple sequence alignment was performedusing Clustal Omega (63) and was visualized with Jalview v.2 (64) (Fig. S1).

Infections. Mice were infected i.v. (0.2 mL) with 107 P. chabaudi pRBCs or 106

PbA into the tail vein and monitored under biosafety level 2 containmentconditions. At predetermined times postinfection, blood was sampled, andblood parasitemia was determined (five fields of >100 erythrocytes werecounted per mouse, and results are expressed as the percentage of para-sitized erythrocytes) following staining of thin blood smears with Diff-Quick.P. berghei-infected mice were similarly monitored for blood parasitemia, butalso for the appearance of neurological symptoms (shivering, tremors, ruf-fled fur, seizures, and paralysis), and were euthanized when reaching clinicalor experimental end points. Survival curves were compared using theMantel–Cox log-rank test.

Plasmodium Metabolic Labeling. Irf8m/m mice were infected i.p. with PbApRBCs and monitored for parasitemia. When the blood parasitemia reached10–15%, blood and was recovered by cardiac puncture in heparinized tubes.

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Fig. 6. CR-1-31B–mediated protection against neuroinflammation is associated with reduced infiltration of immune cells. PbA-infected mice were perfused,and the effect of CR-1-31B on the total number of infiltrating cells, both CD11b+ myeloid cells (A) and TCRb+ lymphoid cells (D), was determined by FACSanalysis. Scatter plots for primary FACS data are shown for CD11b+ myeloid cells (B) and lymphoid cells (E) obtained from noninfected (NI) and PbA-infectedmice treated i.p. with vehicle (Veh) or CR-1-31B. P values were calculated by ANOVA, followed by a Bonferroni posttest. (C and F) Relative mRNA expression(expr.) for representative myeloid (Cebpd) and lymphoid (Gzmb) cell markers in the brain of CR-1-31B–treated and control PbA-infected mice, as determinedby RT-qPCR. Data were normalized to Hprt, used as an internal control, and are expressed relative to noninfected controls (P values were calculated using anunpaired Student’s t test: *P < 0.05; ***P < 0.001).

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Blood was washed three times with 1 vol of cold methionine-free RPMI 1640(ThermoFisher) and resuspended to 5% hematocrit in RPMI 1640 methionine-free medium containing L-glutamine (2 mM), 10% heat-inactivated charcoal-filtered FBS, 0.15 mM hypoxanthine, and 25 mM Hepes. Ninety-six–well platescontaining 5 μL of vehicle or an increasing concentration (from 1 nM to 10 μM)of CHX, CR-1-31B, or CQ were supplemented with 90 μL of PbA-infected blood(or noninfected blood) and incubated at 37 °C for 30 min. Cells were thenlabeled by addition of [35S]-Met (150–225 μCi/mL; PerkinElmer), followed byincubation for 30 min at 37 °C. At the end of the labeling procedure, plateswere immediately put on ice and cold PBS was added. Plates were centrifugedfor 10 min at 500 × g at 4 °C, supernatant was removed, and erythrocytepellets were lysed by addition of 40 μL of lysis buffer (1× PBS, 0.15% saponin,and 0.02% sodium deoxycholate), followed by incubation for 15 min 4 °C withagitation. Twenty microliters of lysate was used for TCA precipitation, andradioactivity was determined by scintillation counting. Also, 5 μL of one rep-licate was analyzed on 10% SDS/PAGE; the gels were treated with En3Hance(PerkinElmer), dried, and exposed to Kodak BioMax XAR Film for 36 h at 20 °C.

Drugs and Drug Treatments. CQ hydrochloride (Sigma) was prepared in PBS,while Art (artesunate; a gift from Dafra Pharmaceuticals, Turnout, Belgium)was prepared fresh in 5% sodium bicarbonate. Asymmetrical synthesis of CR-1-31B and (−)-SDS-1-021 using biomimetic kinetic resolution of chiral, race-mic aglain ketone precursors was performed as previously published (38, 65)followed by amide formation (39). Purity of the rocaglate derivativesCMLD010513 [(−)-CR-1-31B] and CMLD011604 [(−)-SDS-1-021] was assessedusing UPLC-MS-UV-ELSD analysis (Fig. S2). A Waters Acquity UPLC instrument(with a binary solvent manager, single quadrupole (SQ) mass spectrometer,and Waters 2996 photodiode array detector) and an ELSD instrument wereused with an Acquity UPLC BEH C18 1.7-μm column. For the analysis, a sol-vent gradient of 10–90% acetonitrile in water over 2 min, followed by 1 minof 90% acetonitrile, was employed.

For drug treatments, CR-1-31B and (−)-SDS-1-021 were solubilized in 100%DMSO and freshly diluted in sterile PBS to the required concentration with10% DMSO. Vehicle-treated mice received PBS containing 10% DMSO only.The antimalarial activity of CR-1-31B and (−)-SDS-1-021 was tested accordingto experimental protocols we have previously described (57).

Evans Blue Dye Extravasation. At day 6 postinfection with PbA, 24 h afterreceiving a single dose (0.5 mg/kg) of CR-1-31B, groups of mice were injectedi.v. with 0.2 mL of 1% Evans Blue dye (E2129; Sigma) prepared in sterile PBS.The dye was allowed to circulate for 1 h, and mice were then exsanguinatedby cardiac puncture and perfused with PBS containing 2 mM EDTA. Brainswere excised, weighed, imaged, and incubated with 1 mL of dimethylformamide for 48 h to extract Evan’s Blue dye from the tissues. The opticaldensity of 50 μL of dimethyl formamide that received brain Evan’s Blue dyewas measured at 620 nm, and measurements were converted into micro-grams of dye extravasated per gram of tissue by interpolation of a standardcurve from serial dilutions of Evan’s Blue dye in dimethyl formamide.

Leukocyte Infiltration in the Brain. Six days postinfection with PbA, groups ofmice that received single-dose treatment on day 5 were exsanguinated andperfused as above. Brains were harvested; Dounce-dissociated; and ho-mogenized in RPMI media containing 0.5 mg/mL collagenase D (Gibco LifeTechnologies), 0.01 mg/mL DNase I (Roche), and 2 mM EDTA. Infiltrating cellswere separated on a 34% Percoll solution. Cells were counted and stained forflow cytometry analyses with the following: Zombie AquaDye-V500 (BioLegend),anti–CD4-peridinin chlorophyll protein complex (PerCP)-Cyanine5.5 (clone RM4-5; eBioscience), anti–CD8α-eFluor450 (clone 53-6.7; eBioscience), anti–TCR-β–phycoerythrin (PE) (clone H57-597; eBioscience), anti–B220-FITC (RA3-6B2;eBioscience), CD69-BV605 (H1.2F3; BD Bioscience), CD44-PE-Cy7 (clone IM7;eBioscience), anti–CD11b-eFluor450 (clone M1/70; eBioscience), anti–CD11c-PerCP-Cyanine5.5 (clone N418; eBioscience), and anti–Ly6G-FITC (clone 1A8;BioLegend). Flow cytometry was performed on a BD LSRFortessa (BD Bioscience)and analyzed using FlowJo software v.10. A fraction of total brain cells, beforePercoll separation, were lysed, and total RNA was prepared using RNeasycolumns (Qiagen). cDNA was synthesized with Moloney murine leukemiavirus reverse transcriptase (Thermo Fisher Scientific). Myeloid and lymphoidmarkers, as well as IFN-stimulated gene expression levels, were assessed byqPCR using SsoAdvanced SYBR-Green Supermix (BioRad), and gene ex-pression was normalized to Hprt; oligonucleotide sequences were describedpreviously (55). Fold changes in gene expression were expressed relative tononinfected mice.

Preparation of BMDMs. BMDMs were differentiated from bone marrow iso-lated from the femur and tibia of 8- to 16-wk-old C57BL/6 mice, as describedpreviously (45). Briefly, bone marrow cells were cultured in Dulbecco’smodified Eagle’s medium (DMEM; Wisent) containing 10% heat-inactivatedFBS, antibiotics, and 20% L cell-conditioned medium (LCCM). The cells weresupplemented with 10% LCCM 4 d later, and at day 6, cells were harvestedby gentle washing of the monolayer with PBS-citrate. Cells were plated intissue culture-grade six-well dishes in DMEM containing 10% FBS and 20%LCCM, and were used the next day.

Molecular Biology. A codon-optimized variant of PfeIF4A (XM_001348793.1)for increased bacterial expression was synthesized by Genscript and clonedinto pET15b utilizing NdeI and BamHI restriction sites. The resulting plasmidwas transfected in BL21(DE3) codon+ E. coli cells, and the overexpressedprotein was purified using nickel affinity (HiTrap IMAC FF; GE Healthcare),anion exchange (HiTrap Q HP; GE Healthcare), and gel filtration (Superdex200 10/300 GL; GE Healthcare) chromatography. Fractions containingPfeIF4A were pooled and concentrated using Amicon Ultra centrifugal filterunits. The construct pET15b/eIF4AI for expression of murine eIF4AI has beendescribed previously (66).

The ability of different compounds to affect binding and retention ofeIF4A to RNA templates was determined using purified recombinant proteinsand [32P]-labeled RNA templates as previously described (31). To monitor theeffect of CR-1-31B on the proinflammatory response of primary macro-phages, whole-cell protein extracts were prepared and analyzed by SDS/PAGE. Immunoblotting was performed using primary antibodies directedagainst IRF1 (sc-640), STAT1 (sc-591), and GBP2 (sc-10588) (all from SantaCruz Biotechnology); CXCL10 (R&D Systems); and B-Actin (EMD Millipore),and it was revealed using IR680 or IR800-conjugated secondary antibodies(Li-Cor). Protein band intensities were measured using Image Studio Litesoftware (Li-Cor) and normalized against B-Actin protein level.

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ACKNOWLEDGMENTS. We thank Susan Gauthier, Geneviève Perreault, andPatrick Sénéchal for technical assistance. This work was supported by a researchgrant (to P.G.) from the Canadian Institutes of Health Research (CIHR) (Founda-tion Grant). J.P. and P.G. are supported by a James McGill Professorship salaryaward. D.L. is supported by fellowships from the Fonds de recherche santé

Québec, the CIHR Neuroinflammation training program. J.P. is supported byCIHR Research Grant FDN-148366. M.S. is supported by a CIHR Foundation grant.J.A.P. is supported by NIH Grant R35 GM118173. Work at the Boston UniversityCenter for Molecular Discovery is supported by Grant R24 GM111625. K.C.K. wassupported by a CIHR Foundation Grant and the Canada Research Chair program.

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