Serge Rivest Neuroscience Laboratory

CHU de Québec Research Center, Department of Molecular Medicine, Laval University, 2705 Laurier Blvd.,

Québec, Canada  

The Amyloid Code!

Tg APP23 mouse!

Microglial Migration !

In  2013,  Neuron  was  celebra4ng  25  years  of  publishing  exci4ng  neuroscience  and  this  paper  has  been  selected  as  the  most  influen4al  paper  for  year  2006  (hCp://www.cell.com/neuron/twenty-­‐five-­‐years).    

Microglia of bone marrow origin migrate towards endogenous β-amyloid of APPSwe/PS1 mice.

5 mo

6 mo

9 mo

Alzheimer Mouse

Mutation in the APP and PS1 genes

Defect in M1, not

M2!!

CCR2 KO!

Lentivirus-expressing CCR2 in BMSCs rescues!the cognitive deficit in APP/CCR2 KO mice!

CCR2-gene!Delivery

in!Cells of the bone

marrow in CCR2 KO!APP mice!!

The deposition of Aβ in APP/PS1/Cx3cr1 mice is age- and vessel-dependent

Different patterns of Aβ deposition in cortical arteries and veins

Monocytes are selectively attracted to small Aβ aggregates in veins

   

APP/PS1/Cx3cr1gfp/+  (5  mo)   Cx3cr1gfp/+  APP/PS1/Cx3cr1gfp/+  (9  mo)  

Monocytes are selectively attracted to small Aβ aggregates in veins

   

Crawling monocytes can internalize Aβ and circulate back to the bloodstream

Crawling monocytes can internalize Aβ and circulate back to the bloodstream

Deposition of vascular Aβ is dynamic in young mice and correlates with monocyte crawling

Crawling cells inside Aβ+ veins are mainly Ly6Clow monocytes

Reduction of Ly6Clow monocytes increases Aβ deposition

Conclusions

•  Ly6Clo  monocytes  are  a?racted  to  and  crawl  selecBvely  onto  Aβ-­‐posiBve  veins  •  Monocytes  take  up  Aβ  and  circulate  back  to  the  bloodstream  •  Aβ  deposiBon  and  eliminaBon  by  monocytes  is  dynamic  in  veins  of  young  APP/PS1  mice  •  AblaBon  of  Ly6Clo  monocytes  in  APP/PS1  mice  increases  Aβ  load    

Neuron! Yong-Rivest!

Toll-like receptor 4 stimulation with the detoxifiedligand monophosphoryl lipid A improves Alzheimer’sdisease-related pathologyJean-Philippe Michauda, Maxime Halléb, Antoine Lamprona, Peter Thériaulta, Paul Préfontainea, Mohammed Filalia,Pascale Tribout-Joverb, Anne-Marie Lanteigneb, Rachel Jodoinb, Christopher Cluffc, Vincent Brichardd, Rémi Palmantierd,Anthony Pilorgetb, Daniel Larocqueb,1, and Serge Rivesta,1

aNeuroscience Laboratory, Department of Molecular Medicine, Centre Hospitalier Universitaire de Québec Research Center, Laval University, Québec City, QC,Canada G1V 4G2; bGlaxoSmithKline Vaccines, Laval, QC, Canada H7V 3S8; cGlaxoSmithKline Vaccines, Hamilton, MT 59840; and dGlaxoSmithKline Vaccines,B-1330 Rixensart, Belgium

Edited by Shizuo Akira, Osaka University, Osaka, Japan, and approved December 19, 2012 (received for review September 1, 2012)

Alzheimer’s disease (AD) is the most common cause of dementiaworldwide. The pathogenesis of this neurodegenerative disease,currently without curative treatment, is associated with the accu-mulation of amyloid ! (A!) in brain parenchyma and cerebral vas-culature. AD patients are unable to clear this toxic peptide, leadingto A! accumulation in their brains and, presumably, the pathologyassociated with this devastating disease. Compounds that stimulatethe immune system to clear A! may therefore have great therapeu-tic potential in AD patients. Monophosphoryl lipid A (MPL) is anLPS-derived Toll-like receptor 4 agonist that exhibits unique immu-nomodulatory properties at doses that are nonpyrogenic. We showhere that repeated systemic injections of MPL, but not LPS, signif-icantly improved AD-related pathology in APPswe/PS1 mice. MPLtreatment led to a significant reduction in A! load in the brain ofthese mice, as well as enhanced cognitive function. MPL induceda potent phagocytic response by microglia while triggering a mod-erate inflammatory reaction. Our data suggest that the Toll-like re-ceptor 4 agonist MPL may be a treatment for AD.

innate immunity | microglial cells | monocytes | phagocytosis |inflammation

Alzheimer’s disease (AD) is a neurodegenerative pathologycharacterized by the accumulation of amyloid beta (Aβ) and

neurofibrillary tangles in the brain parenchyma (1). Inflammation,which occurs in parallel with the progression of the disease, isfeatured by the production of cytokines by activatedmicroglia. Therole of these cells in the pathogenesis of AD remains unclear andis an area of active investigation. Whereas chronic activation ofmicroglial cells by Aβ can trigger the exaggerated release of cyto-kines and neurotoxic mediators that could be detrimental to neu-rons, microglia can also clear Aβ via increased phagocytosis andproteolytic degradation, which may be neuroprotective (2).Toll-like receptors (TLRs) on the surface of microglial cells have

been shown to bind Aβ, which triggers downstream intracellularsignaling cascades (3, 4). Microglia deficient in TLR2, TLR4, orthe coreceptor CD14 are not activated by Aβ and do not exhibita phagocytic response (5). Transgenic AD mice lacking TLR4 havemarkedly elevated levels of diffuse and fibrillar Aβ (3). Furthermore,stimulation of microglial cells with TLR2-, TLR4-, or TLR9-specificagonists accelerates Aβ clearance both in vitro and in vivo (3, 6, 7).

Monophosphoryl lipid A (MPL) is a chemically detoxified lipidAmoiety derived from Salmonella minnesota R595 LPS (8). ThisTLR4 ligand is at least 100-fold less pyrogenic than LPS yet main-tains many of the immunomodulatory properties of LPS (9). Im-portantly, MPL is safe in humans and has been administered tomillions of patients as a component of several vaccine formulationssuch as the Cervarix vaccine (10). We investigated herein thechronic use of the nonpyrogenic TLR4 agonist MPL and comparedit with a strong TLR4 ligand (LPS) in a mouse model of AD.Although the therapeutic potential of innate immune activa-

tion for AD is being evaluated in preclinical models, this concept

has not been tested in humans. We propose that the age-relateddefects in immune cell function (11) commonly found in agingdiseases such as AD (12) can be reconciled with a prophagocyticphenotype, yet mildly proinflammatory, which may lead to animproved clearance of Aβ.Our data demonstrate that chronic, systemic administration of

MPL ameliorates AD-like pathology by decreasing the cerebralAβ load through the stimulation of the phagocytic capacity ofinnate immune cells.

ResultsMPL Drives a Distinct TLR4 Stimulation from LPS. MPL is derivedfrom the LPS of theGram-negative bacteria SalmonellaminnesotaR595 by three main chemical modifications: (i) elimination of thecore oligosaccharide, (ii) hydrolysis of the 1-phosphate from thereducing end glucosamine, and (iii) removal of the acyl chain fromthe 3-position of the disaccharide (Fig. 1A and B). The absence ofthe 1-phosphate on the MPL molecule was suggested to weakenthe dimerization of TLR4/MD2 (myeloid differentiation factor-2)(13). This presumably induces a structural change in the TLR4receptor complex that alters the recruitment of the adaptor pro-teins to the intracellular domain (13). Such a structural changemay account for the distinct signaling properties of MPL, whichpredominantly activates the TLR4-TRAM (TRIF-related adap-tor molecule)-TRIF (TIR-domain-containing adaptor proteininducing IFN-β) pathway over the more proinflammatory TLR4-MAL (MyD88-adaptor-like protein)-MyD88 (myeloid differen-tiation primary-response protein 88) signaling pathway (14). Thisdifferential use of intracellular adaptor proteins may be key toexplaining the distinct effects observed after exposure of cells toMPL or LPS.To characterize the ligand–receptor interaction of MPL with

the TLR4 receptor complex we used the HEK293 cell linetransfected with TLR4, MD2, and CD14 genes, as well as anNF-κB and AP-1 reporter system. At the highest concentrationof MPL tested (20 μg/mL), the activation of NF-κB and AP-1 wasat a level comparable to a 200-fold lower concentration of LPS(0.1 μg/mL) (Fig. 1C). A neutralizing antibody directed againstTLR4 inhibited the response to MPL. Incubating TLR2-trans-fected HEK293 cells with up to 2.5 μg/mL of MPL did not induceany activation of NF-κB and AP-1 (Fig. 1D), indicating that the

Author contributions: J.-P.M., A.L., A.P., D.L., and S.R. designed research; J.-P.M., M.H., A.L.,P.T., P.P., M.F., P.T.-J., A.-M.L., and R.J. performed research; J.-P.M., M.H., A.L., P.P., and D.L.analyzed data; and J.-P.M., M.H., A.L., C.C., V.B., R.P., A.P., D.L., and S.R. wrote the paper.

Conflict of interest statement: M.H., P.T.-J., A.-M.L., R.J., C.C., V.B., R.P., A.P., and D.L. areemployees of GlaxoSmithKline Vaccines. This research was supported in part byGlaxoSmithKline Vaccines.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. E-mail: daniel.a.larocque@gsk.com orserge.rivest@crchul.ulaval.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1215165110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1215165110 PNAS Early Edition | 1 of 6

NEU

ROSC

IENCE

Comparison  of  LPS  and  MPL  extracted  from  Salmonella  minnesota  R595.    

(i)  eliminaBon  of  the  core  oligosaccharide    (ii)  hydrolysis  of  the  1-­‐phosphate  from  the  reducing  end  glucosamine  (iii)  removal  of  the  acyl  chain  from  the  3-­‐posiBon  of  the  disaccharide    

TLR4-­‐specific  ac=va=on  of  NF-­‐κB  and  AP-­‐1  in  HEK  293  cells    

TLR4-­‐MD2-­‐CD14   TLR2  

Phosphoryla=on  kine=cs  of  key  TLR  pathway  of  MPL  vs  LPS  in  microglia  

MPL  induces  a  low  inflammatory  response  in  microglia  

MPL  s=mulates  ac=n  reorganiza=on  

Phagocytosis  of  fluorescent  E.coli  beads  and  Aβ  oligomers  

MPL  upregulates  Scavenger  receptors-­‐A  in  microglia  

In  vivo  uptake  of  Aβ  oligomers  in  monocytes  

Low  immune  ac=va=on  following  MPL  injec=on  

MPL  triggers  only  a  weak  TLR2  mRNA  induc=on  in  the  brain  

MPL  triggers  the  expansion  of  blood  monocytes  in  mice    

APPswe/PS1  mice  treatment  

-­‐  3  month-­‐old  APPswe/PS1  mice    -­‐  12  consecu=ve  i.p.  injec=ons  once  a  week    -­‐  MPL  (50  μg,  130  μL)        LPS  (3,5  μg,  130  μL)        PBS  (130  μL)      -­‐  Pathology  analysis  at  6  month-­‐old  

MPL  treatment  reduces  Aβ  plaques    

MPL  treatment  reduces  soluble  Aβ  

MPL  treatment  reduces  cogni=ve  deficits  in  APPswe/PS1  mice  

Conclusions  

-­‐  MPL  induces  a  potent  phagocy=c  response  in  microglia  and  monocytes  (p38  pathway  and  SR-­‐A)  

-­‐  MPL  triggers  a  low  inflammatory  response  -­‐  MPL  reduces  Aβ  loads  and  restricts  cogni=ve  deficits  of  APPswe/PS1  mice.  

Neuron! Yong-Rivest!

Activation of systemic innate immune cells is a very efficient approach to clear vascular and brain amyloid load and prevent cognitive decline in Alzheimer's disease. This may also be the case for other brain diseases associated with the accumulation of toxic extracellular proteins (SOD1, alpha-Syn, …).

Who did the work? Marc-André Bellavance, M.Sc. Dr. Alain Simard Dr. Antoine Lampron Dr. Gaëlle Naert Jean-Philippe Michaud, M.Sc. Paul Préfontaine. M.Sc. Dr. Maxime Hallé (GSK) Dr. Daniel Larocque (GSK)

$$$ CIHR, CRC, Neuroscience Canada, Brain Repair Program (Dr. Wee Yong, PI), MS Canada Team Grant (Dr. Peter Stys, PI), Canadian Stroke Network, GSK-Vaccines.

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