implication du remodelage de l’unité neurovasculaire dans la … · 2018-04-24 · iii résumé...
TRANSCRIPT
Implication du remodelage de l’unité
neurovasculaire dans la maladie d’Alzheimer : L’hypoperfusion cérébrale et le système de l’activateur
tissulaire du plasminogène
Mémoire
Maude Bordeleau
Maîtrise en Neurobiologie
Maître ès sciences (M.Sc.)
Québec, Canada
© Maude Bordeleau, 2016
iii
Résumé
L’unité neurovasculaire (NVU) est centrale dans l’élimination de la β-amyloïde dont l’accumulation
promeut le développement de la maladie d’Alzheimer (AD). Suivant une perturbation vasculaire, le
bris ou l’altération de la barrière hématoencéphalique induit le remodelage de la NVU. Par exemple,
les cellules endothéliales sécrètent l’activateur tissulaire du plasminogène (t-PA), ce qui module les
cellules composant la NVU. C’est pourquoi, nous nous sommes intéressés à ce remodelage dans la
AD en étudiant l’effet de l’hypoperfusion cérébrale chronique sévère (SCCH) et de l’administration
du t-PA. Suite à la SCCH, les souris développant la AD, APPswe/PS1, démontrent un déclin
cognitif plus important causé par un dysfonctionnement des microglies. En contre partie, nous
avons observé une amélioration des fonctions cognitives des APPswe/PS1 suite à l’injection
systémique du t-PA qui induit l’activation des microglies via la protéine apparentée au récepteur des
protéines de faibles densité, LRP1, et promeut l’élimination de l’Aβ. Ainsi, nos résultats démontrent
que le remodelage de la NVU peut aggraver la pathogenèse, mais également fournir des pistes de
traitement.
v
Abstract
Brain remodeling by the neurovascular unit (NVU) has gain interest in disease such as Alzheimer’s
disease (AD). Following vascular perturbation, NVU go through remodeling due to disruption or
alteration of brain-blood barrier. One of the molecule inducing remodeling is the tissue-
plasminogen activator (t-PA) released by endothelial cells. In fact, t-PA can act both as an enzyme
and a cytokine. Thus, we studied the effect of vascular perturbation and t-PA system in AD. By
developing a new model of a severe chronic cerebral hypoperfusion (SCCH), we demonstrate that
SCCH aggravates memory loss in AD mice, APPswe/PS1, due to microglia dysfunction. Indeed,
low glucose environment lowers microglia’s activity and phagocytosis capacity. On the other hand,
systemic administration of t-PA improves cognition as well as decreases amyloid burden in
APPswe/PS1. Acting as a cytokine, rt-PA binds LRP1 which induces microglia’s activation and
promotes amyloid elimination. These data suggest that NVU remodeling occurring in AD may
participate in the disease pathogenesis and provide new insight of treatment, such as rt-PA.
vii
Table des matières
Résumé ....................................................................................................................................... iii
Abstract ....................................................................................................................................... v
Table des matières ..................................................................................................................... vii
Liste de tableaux ......................................................................................................................... xi
Liste de figures ......................................................................................................................... xiii
Abréviations .............................................................................................................................. xv
Remerciements ......................................................................................................................... xix
Avant-propos ............................................................................................................................ xxi
1. Introduction ......................................................................................................................... 1
1.1. Maladie d’Alzheimer ......................................................................................... 1
1.1.1. Tau ...................................................................................................................... 3
1.1.2. Amyloïde ............................................................................................................. 4
1.1.2.1. Formation, élimination et dégradation de l’amyloïde ..................................... 4
1.1.2.2. Agrégation de l’amyloïde ................................................................................ 7
1.1.2.3. Hypothèse de la cascade amyloïde .................................................................. 7
1.1.3. Hypothèse vasculaire .......................................................................................... 9
1.2. Facteurs de risque de la maladie d’Alzheimer ................................................... 9
1.2.1. Génétiques ........................................................................................................... 9
1.2.2. Obésité .............................................................................................................. 10
1.2.3. Diabète .............................................................................................................. 10
1.2.4. Troubles vasculaires .......................................................................................... 11
1.3. Hypoperfusion ................................................................................................. 11
1.3.1. Régulation du flux sanguine et maladie d’Alzheimer ....................................... 11
1.3.2. Oligémie versus ischémie ................................................................................. 12
1.4. Système de l’activateur tissulaire du plasminogène ........................................ 13
1.4.1. Fonction du système de l’activateur tissulaire du plasminogène ...................... 13
1.4.2. Système de l’activateur du plasminogène et maladie d’Alzheimer................... 15
1.5. Unité neurovasculaire ...................................................................................... 16
1.5.1. Fonction de l’unité neurovasculaire .................................................................. 17
1.5.2. Remodelage de l’unité neurovasculaire et maladie d’Alzheimer ...................... 19
1.6. Hypothèses et objectifs .................................................................................... 20
2. Severe chronic cerebral hypoperfusion induces microglial dysfunction leading to memory
loss in APPswe/PS1 mice .................................................................................................. 23
2.1. Résumé ............................................................................................................ 24
2.2. Abstract ............................................................................................................ 24
viii
2.3. Introduction ...................................................................................................... 25
2.4. Material and methods ....................................................................................... 26
2.4.1. Animals with severe chronic cerebral hypoperfusion ....................................... 26
2.4.2. Behavior analysis .............................................................................................. 28
2.4.2.2. Two-object novel object recognition ............................................................. 28
2.4.2.3. Asymmetry cylinder test ................................................................................ 29
2.4.2.4. Open field ...................................................................................................... 29
2.4.3. Soluble Aβ1-40 and soluble Aβ1-42 ELISA .......................................................... 29
2.4.4. Immunofluorescence staining ............................................................................ 30
2.4.5. Western blot analysis ......................................................................................... 30
2.4.6. Flow Cytometry ................................................................................................. 31
2.4.7. Immunohistochemistry ...................................................................................... 32
2.4.8. Nissl body staining ............................................................................................ 32
2.4.9. Fluoro-Jade B staining ....................................................................................... 33
2.4.10. In vitro experiments ........................................................................................... 33
2.4.10.1. Cell culture................................................................................................... 33
2.4.10.3. Griess assay ................................................................................................. 34
2.5. Results .............................................................................................................. 35
2.5.1. SCCH worsen memory impairment in APPswe/PS1 mice without affecting
motor capacity .................................................................................................. 35
2.5.2. Memory loss in SCCH mice is associated with an increased number of
parenchymal amyloid plaques .......................................................................... 37
2.5.3. SCCH-linked trend towards an increased in the patrolling monocyte population
......................................................................................................................... 37
2.5.4. SCCH disrupts plaque coverage by microglia and alters microglial activation 39
2.5.5. Alteration of microglial function is caused by an impaired glucose metabolism
......................................................................................................................... 40
2.5.6. ERK pathway-dependent decrease in cell survival contributes to memory
impairment in SCCH mice ............................................................................... 42
2.6. Discussion ........................................................................................................ 43
2.8. Acknowledgements ........................................................................................................ 46
2.9. Grant support .................................................................................................................. 46
3. Tissue-plasminogen activator attenuates Alzheimer’s disease-related pathology
development in APPswe/PS1 mice .................................................................................... 47
3.1. Résumé ............................................................................................................. 48
3.2. Abstract ............................................................................................................ 48
3.3. Introduction ...................................................................................................... 49
ix
3.4. Materials and Methods .................................................................................... 50
3.4.1. Animal experiments .......................................................................................... 50
3.4.2. Chimeric mice generation ................................................................................. 51
3.4.3. Tissue collection ................................................................................................ 52
3.4.4. Immunofluorescence staining ........................................................................... 52
3.4.5. IgG and albumin extravasation ......................................................................... 53
3.4.6. Aβ plaques, microglia coverage and Aβ internalization by microglia
quantification ................................................................................................... 53
3.4.7. In situ Hybridization ......................................................................................... 54
3.4.8. Soluble Aβ1–42 Enzyme-Linked Immunosorbent Assay (ELISA) ..................... 54
3.4.9. Brain microvessel isolation ............................................................................... 54
3.4.10. Microglia’s isolation and analysis by flow cytometry ...................................... 55
3.4.11. Protein extraction .............................................................................................. 56
3.4.12. Caseinase and gelatinase activity assays ........................................................... 56
3.4.13. Western blot analysis ........................................................................................ 56
3.4.14. Flow cytometry ................................................................................................. 57
3.4.15. Behavior analysis .............................................................................................. 57
3.4.16. In vitro experiments .......................................................................................... 58
3.4.16.1. Cells culture ................................................................................................. 58
3.4.16.2. Cell stimulation ........................................................................................... 58
3.4.16.3. Cell migration assay .................................................................................... 58
3.4.16.4. Chemotaxis assay ........................................................................................ 59
3.4.16.5. Phagocytosis assay ...................................................................................... 59
3.4.16.6. Griess Assay ................................................................................................ 59
3.4.17. Statistics ............................................................................................................ 60
3.5. Results ............................................................................................................. 60
3.5.1. Activase® rt-PA regimen does not affect blood-brain barrier integrity and
function ............................................................................................................ 60
3.5.2. Activase® rt-PA slows the progression of AD-like pathology and behavioral
deficits in APPswe/PS1 ................................................................................... 62
3.5.3. The enzymatic activity is not responsible of rt-PA-induced clearance of Aβ ... 64
3.5.4. Activase® rt-PA modulates monocyte population phenotypes in a transient
manner ............................................................................................................. 66
3.5.5. The effects of Activase® rt-PA on resident microglia ...................................... 66
3.5.6. Activase® rt-PA enhances BV2 microglial cell mobility and acts as
chemoattractant molecule in a LRP1-dependent manner ................................ 69
x
3.5.7. The effects of Activase® rt-PA on the phagocytic capacity and oxidative stress
cascade in BV2 microglial cells ....................................................................... 69
3.5.8. The effects of Activase® rt-PA on the mobility and the phagocytic capacity of
BV2 microglial cells is independent of its enzymatic activity. ........................ 71
3.6. Discussion ........................................................................................................ 71
3.7. Conclusion ....................................................................................................... 75
3.8. Acknowledgments ............................................................................................ 76
3.9. Funding ............................................................................................................ 76
4. Discussion .......................................................................................................................... 77
5. Conclusions et perspectives ............................................................................................... 87
Références ................................................................................................................................. 89
Annexe – Figures supplémentaires .......................................................................................... 109
xi
Liste de tableaux
Tableau I. Formes mutantes du gène APP découvertes dans les cas familiaux……………........…...2
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Liste de figures
Figure 1.1. Voies de transformation de l’APP………………………………………………………..5
Figure 1.2. Voie d’entrée et d’élimination de la β-amyloïde………………………………..……..6
Figure 1.3. Schéma de l’hypothèse vasculaire de la maladie d’Alzheimer…………………………..8
Figure 1.4. Structure et fonction de l’activateur tissulaire du plasminogène (t-PA)……………….14
Figure 1.5. Représentation de l’unité neurovasculaire et de la microcirculation cérébrale…………17
Figure 2.1. Schema illustrating the SCCH surgery………………………………………………....27
Figure 2.2. SCCH aggravates APPswe/PS1 memory loss…………………………………………36
Figure 2.3. Number of amyloid plaques increase following SCCH without any change in amyloid
burden…………………………………………………………………………………...38
Figure 2.4. A tendency of increased patrolling monocytes is observed following SCCH................39
Figure 2.5.SCCH alters microglial function in APPswe/PS1………………………………………41
Figure 2.6. Low glucose environment alter the activity and the phagocytosis capacity of
microglia...... ……………………………………………………………………………42
Figure 2.7.SCCH lowers ERK1/2 activation………………………………………………………43
Figure. 3.1. Activase® rt-PA administration reduces Aβ aggregates and soluble Aβ1-42 levels in the
brain……………………………………………………………………………………61
Figure. 3.2. Activase® rt-PA administration improves APPswe/PS1 mice cognitive functions…..62
Figure. 3.3. t-PA-associated perivascular proteases are not induced by Activase® rt-PA regimen..63
Figure. 3.4. Chronic Activase® rt-PA administration modulates monocyte subpopulation
frequencies in the blood of APPswe/PS1 mice…………………………………………64
Figure. 3.5. Acute Activase® rt-PA administration modulates monocyte subpopulation frequencies
in the blood of wildtype mice…………………………………………………………65
Figure. 3.6. Chronic Activase® rt-PA administration increases the number of resident microglia-
associated to Aβ plaques and reduces the activation of stress-induced pathways……67
Figure. 3.7. Activase® rt-PA administration does not influence blood-derived monocyte infiltration
into the brain parenchyma of APPswe/PS1 mice………………………………………68
Figure. 3.8. Activase® rt-PA modulates BV2 microglial cell activation in vitro………………….70
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Figure. 3.9. Activase® rt-PA decreases BV2 microglial cell intracellular stress and preserves their
phagocytic capacity……………………………………………………………………72
Figure 4.1. L’effet de l’hypoperfusion cérébrale chronique sévère (SCCH) sur le cerveau des
APPswe/PS1……………………………………………………………………………77
Figure 4.2. L’effet du traitement hebdomadaire de l’Activase ® rt-PA sur le cerveau des
APPswe/PS1……………………………………………………………………………82
Supplementary Figure 2.1. Motricity behavior in APPswe/PS1 after severe chronic cerebral
hypoperfusion (SCCH)………………………………………………109
Supplementary Figure 2.2. SCCH does not alter blood-brain barrier tightness…………………..110
Supplementary Figure 2.3. Absence of infiltred monocytes after SCCH…………………………110
Supplementary Figure 2.4. SCCH seems to atrophy CA3 without neuronal death………………111
Supplementary Figure. 3.1. BBB tightness is preserved after Activase® rt-PA administration…112
Supplementary Figure. 3.2. BBB integrity is preserved after Activase® rt-PA administration…..113
Supplementary Figure. 3.3. Endothelial transporters involved in Aβ transport across the BBB are
not affected following Activase® rt-PA administration……………114
Supplementary Figure. 3.4. Activase® rt-PA regimen does not modulate the brain levels of
synaptophysin………………………………………………………115
Supplementary Figure. 3.5. Acute Activase® rt-PA administration modulates total monocyte
frequency in the blood of APPswe/PS1 mice………………………116
Supplementary Figure. 3.6. Chronic Activase® rt-PA administration does not trigger a sustained
inflammation in the brain of chimeric APPswe/PS1 mice…………116
Figure supplémentaire 4.1. Changement de la déposition vasculaire de l’amyloïde……………117
xv
Abréviations
1VO: 1-vessel occlusion, Ligature unilatérale de l’artère carotide commune
Aβ: Amyloid β-peptide, Protéine β-amyloïde
ABCB1: ATP-binding cassette sub-family B1, Transporteur à cassette liant l’ATP de la sous-
famille B1
ACE: Angiotensin converting enzyme, Enzyme de conversion de l’angiotensine
AD: Alzheimer disease, Maladie d’Alzheimer
AMPA: α-amino-3-hydroxy-5-méthylisoazol-4-propionate
APH-1: Anterior pharynx 1, Protéine du pharynx antérieur défectueux 1
Apo: Apolipoprotéine
APP: Amyloid precursor protein, Protéine précurseure de l’amyloïde
APP-sα: APP-α soluble
APP-sβ: APP-β soluble
APPswe: Gène APP possédant la double mutation familiale Swedish (K670N, M671L)
APPswe/PS1: Souris transgénique double-mutante APP Swedish et PS1 A246E
ATP: Adénosine tri-phosphate
AVC: Accident vasculaire cérébral
BACE-1: β-site APP cleaving enzyme 1, Enzyme 1 de clivage du site β de l’APP
BBB: Blood-brain barrier, Barrière hémato-encéphalique
BCAO: Bilateral common carotid arthery occlusion, Ligature bilatérale des artères
carotides communes
BCAS: Bilateral common carotid arthery stenosis, Sténose bilatérale des artères carotides
communes
BDNF: Brain-derived neurotrophic factor
BSA: Bovin serum albumin, Albumine de serum bovin
CA: Cornu ammonis, Corne d’Ammon
CAA: Cerebral amyloid angiopathy, Angiopathie amyloïde cérébrale
CBF: Cerebral blood flux, Flux sanguin cérébral
CTFα: C-terminal fragment α, Fragment carboxy-terminal α
CTFβ: C-terminal fragment β, Fragment carboxy-terminal β
DAB: Diaminobenzidine
DAPI: 4,6-diamindino-2-phenylindone
xvi
DMEM: Dulbeco’s modified eagle medium, Milieu Eagle modifié de Dulbeco
DMEM High: DMEM contenant 4500mg/L de glucose
DMEM Low: DMEM contenant 1000mg/L de glucose
EDTA: Ethylenediaminetetraacetic acid, Acid éthylène diamine-tétraacétatique
ECL: Enhanced chimiluminescence solution
EGF: Epidermal growth factor, Facteur de croissance de l’épiderme
ERK: Extracellular signal-regulated kinase, Kinase régulée par les signaux
extracellulaires
ELISA: Enzyme-linked immunosorbent assay, Essai d’immuno absorption enzymatique
FACS: Fluorescent-activated cells sorting, Tri de cellules activées par fluorescence
FBS: Fetal bovine serum, Sérum de veau fœtal
FDA: Food and Drug Association
FJB: Fluoro-jade B
FTD: Frontotemporal dementia, Démence fronto-temporale
GFP: Green fluorescent protein, Protéine fluorescente verte
GLUT1: Glucose transporter 1, Transporteur du glucose 1
HBSS: Hank’s balanced salt solution, Solution saline équilibrée de Hank
HIF-1α: Hypoxia-inducible factor 1α, Facteur de transcription 1α induit par l’hypoxie
HRP: Horseradish peroxidase, Peroxidase de raifort
IDE: Insulin degrading enzyme, Enzyme de dégradation de l’insuline
IgG: Immunoglobuline G
IL: Interleukine
KPBS: Potassium phosphate buffered saline, Saline tamponée au potassium et phosphate
LDL: Low-density lipoprotein, Lipoprotéine à faible densité
LPS: Lipopolysaccharide
LRP: LDL receptor-related protein, Protéine apparentée au récepteur des LDLs
LTD: Long-term depression, Dépression à long-terme
LTP: Long-term potentiation, Potentialisation à long-terme
MAPK: Mitogen-activated protein kinase, Protéine kinase activée par mitose
MCI: Mild cognitif impairment, Trouble cognitif léger
mGluR5: Metabotropic glutamate receptor 5 Récepteur métabotropique du glutamate 5
MMP: Matrix metalloproteinase, Métalloprotéase matricielle
MPL: Monophosphoryle lipide A
MVs: Microvessels
xvii
ND: No cognitif deficit
NEP: Néprilysine
NGF: Nerve growth factor, Facteur de croissance des nerfs
NMDA: N-méthyl-D-aspartate
NVU: Neurovascular unit, Unité neurovasculaire
OMS: Organisation Mondiale de la Santé
PAI-1: Plasminogen activator inhibitor-1, Inhibiteur-1 des activateurs du plasminogène
PBS: Phosphate buffered saline, Saline tamponnée au phosphate
PDGF-CC: Platelet-derived growth factor-CC, Facteur de croissance CC dérivé des plaquettes.
PEN-2: Preseniline enhancer 2, Protéine activatrice de la préséniline 2
PET: Positron emission tomography, Tomographie à émission de positrons
PFA: Paraformaldéhyde
PgP: P-glycoprotéine
PrP: Prion protein, Protéine prionique
PS: Préséniline
RAGE: Receptor for advanced glycation endproducts, Récepteur des produits finaux de
glycosylation avancée
RT: Room temperature
rt-PA: Recombinant-tisssue-type plasminogen activator, Activateur tissulaire du
plasminogène recombinant
SAPK/JNK: Stress-activated protein kinases /Jun amino-terminal kinases, Protéine kinase active
par le stress / Kinase c-Jun N-terminal
SCCH: Severe chronic cerebral hypoperfusion, Hypoperfusion cérébrale chronique sévère
S.D.: Standard deviation, Écart-type
SD: Severe cognitif deficit
SDS-PAGE: SDS-polyacrylamide gel electrophoresis, Gel d’électrophorèse SDS-polyacrylamide
S.E.M.: Standard error of the mean, Écart-type de la moyenne
sLRP: LRP soluble
SNC: Système nerveux central
TLR: Toll-like receptor, Récepteur de type Toll
t-PA: Tissue-type plasminogen activator, Activateur tissulaire du plasminogène
xix
Remerciements
Au cours de ces deux dernières années, j’ai côtoyé un ensemble de gens qui m’ont offert le soutien
nécessaire pour réussir et aboutir dans mes projets entrepris. Je tiens donc à souligner leur
contribution qui a été très marquante.
Tout d’abord, je voudrais remercier Serge Rivest, Ph.D., qui m’a offert la chance de travailler au
sein de son équipe, ainsi que Frédérique Calon, Ph.D., et Denis Soulet, Ph.D., qui ont accepté de
faire partie de mon comité d’encadrement. Tous m’ont offert de précieux conseils en tant que
scientifique. Ils m’ont guidé pour l’avancement de mes projets vers la bonne direction. Surtout au
cours des derniers mois, Dr. Rivest a toujours été prêt pour discuter des derniers détails et ce,
malgré son horaire chargé. Je voudrais également souligner l’énorme contribution d’Ayman ElAli,
Ph.D., en tant superviseur de projet. Servant de guide et de collègue, Dr. ElAli m’a offert un soutien
continu pour que je retire la meilleure expérience qui soit de ma maîtrise. Il a été sans conteste un
mentor important qui a permis l’avancement de mes projets et stimulé ma curiosité scientifique.
Au cours de ma maîtrise, j’ai également eu la chance de profiter de l’expertise de plusieurs
personnes qui m’ont conseillé lorsque j’avais des interrogations. Nataly Laflamme, M.Sc., Marie-
Michèle Plante, M.Sc., Paul Préfontaine, M.Sc., Antoine Lampron, Ph.D., Jean-Philippe Michaud,
Ph.D., Peter Thériault, M.Sc., et Audrey LeBehot, Ph.D., m’ont tous offert leur avis lorsque je leur
posais une panoplie de questions. J’ai également eu l’opportunité de connaitre brièvement Marc-
André Bellavance, Ph.D., et Antoine Larochelle, M.Sc.
Sur un plan plus personnel, je voudrais remercier Yannick Tremblay, B.Sc., Édith Godbout-Miville,
B.Sc., Catherine Fontaine-Lavallée, B.Sc., Catherine Gilbert, B.Sc., André-Pascal Roy, B.Sc.,
Prenitha Innatious, M.Sc., avec qui j’ai discuté de science comme de tout et n’importe quoi.
Comparse dans l’acheminement de nos travails respectifs, discuter avec eux a toujours allégé la
morosité d’une série de résultats négatifs. Je dois également souligner le support de ma famille et
amis qui m’ont aidé à décrocher lorsque j’en avais besoin ce qui m’a permis d’être toujours positive
dans mon activité de recherche.
Finalement, un dernier merci pour toutes ces personnes qui, sans le savoir, m’ont permis d’avoir
une expérience dont je me souviendrai et de m’apprendre tant de choses autant sur le plan
scientifique que personnel. Merci.
xxi
Avant-propos
L’article intitulé Severe chronic cerebral hypoperfusion induces microglial dysfunction leading to
memory loss in APPswe/PS1 mice est en revision pour le journal Oncotarget. Cet article représente
le second chapitre du mémoire. Lors de ce projet, j’ai effectué l’intégralité des expériences mise à
part le comportement qui a été réalisé par Mohammed Filali. J’ai également accompli l’analyse des
résultats, puis interprété ceux-ci en discutant avec Ayman ElAli. Suite à la discussion scientifique,
j’ai rédigé intégralement l’article scientifique qui a ensuite été corrigé par Ayman ElAli et Serge
Rivest. Le projet lui-même a été conçu et encadré par Ayman ElAli et Serge Rivest.
L’article intitulé Tissue-plasminogen activator attenuates Alzheimer’s disease-related pathology
development in APPswe/PS1 mice a été publié en ligne dans Neurospychopharmacology le 9
septembre 2015 (ElAli, A., Bordeleau, M., Thériault, P., Filali, M., Lampron, A. et Rivest, S.
Neuropsychopharmacology. doi:10.1038/npp.2015.279.). Celui-ci constitue le troisième chapitre de
ce mémoire. Dans ce projet, j’ai effectué l’ensemble de l’étude in vitro décrivant le mécanisme
d’action du t-PA, l’analyse et l’interprétation des résultats, en plus de la rédaction du matériel et
méthode correspondant. J’ai également coupé les tissus et effectué les marquages sur les tissus
(FJB, hybridation in situ, immunofluorescence). Peter Thériault a effectué l’expérience de FACS,
aider à l’analyse et rédiger la partie matériel et méthode correspondante. Mohammed Filali ont
effectué les tests de comportement, de même que la rédaction du matériel et méthode associée.
Ayman ElAli a effectué le reste des mannipulation in vivo, ainsi de l’analyse des résultats et
l’interprétation de l’ensemble des résultats in vivo. L’article a été principalement écrit par Ayman
ElAli, puis corrigé par l’ensemble des co-auteurs. Le projet lui-même a été conçu et encadré par
Ayman ElAli et Serge Rivest
1
Chapitre 1
1. Introduction
1.1. Maladie d’Alzheimer
La maladie d’Alzheimer (AD) est un désordre neurodégénératif se développant au cours d’une vie
et représentant la forme de démence la plus répandue (1). Au cours des dernières années,
L’Organisation Mondiale de la Santé (OMS) a estimé que le nombre de gens atteints par la AD
s’élève à 30 millions ; nombre qui devrait tripler au cours des prochaines décennies (2,3). Chez la
population âgée de plus de 85 ans, 1 personne sur 3 développe des symptômes de la AD (3).
Celle-ci a été diagnostiquée pour la première fois, en 1907, par Aloïs Alzheimer. Il avait alors
observé la présence de plaques denses dans la parenchyme, appelé plaques séniles, et de
neurofibrilles (4). Il est maintenant connu que ces plaques et neurofibrilles sont respectivement
entraînées par la déposition du peptide β-amyloïde (Aβ) (5,6), dérivé de la protéine précurseure de
l’amyloïde (APP), et l’hyperphosphorylation de tau, une protéine associée aux microtubules, qui
forme des filaments (7,8). Ces phénomènes moléculaires entraînent principalement un
dysfonctionnement de la capacité mnésique qui s’instaure graduellement. Braak et Braak ont
distingué six paliers (I à VI) à cette progression selon les régions du cerveau touchées. Les premiers
changements neuropathologiques débutent dans le cortex entorhinal (I-II), puis s’étendent aux
régions limbiques (III-IV) et, aux aires associatives temporales, pariétales et frontales du néocortex
(V-VI) (9,10). Récemment, un stade préclinique de la AD a été identifié. Celui-ci se décrit par un
trouble cognitif léger (MCI) (11) se manifestant quelques années avant le diagnostic de démence et
étant notamment associés avec plusieurs problèmes causé par le remodelage du cerveau (Section
1.5.2 – Remodelage de l’unité neurovasculaire et maladie d’Alzheimer) (1). Durant cette
période, le patient présente des troubles cognitifs, mais trop légers pour interférer dans sa vie de
tous les jours (1). Les premières manifestations cliniques de la AD, soit post-diagnostic, démontrent
généralement une dysfonction de la mémoire de travail et de la mémoire sémantique, en plus de la
sensibilité aux distractions. Il a été également reporté que, lors de la phase initiale, les malades
d’Alzheimer peuvent être dépressifs ou apathiques. Dans un stade plus avancé, ceux-ci peuvent
devenir confus, désorientés et avoir des comportements anormaux (1) associés à une perturbation du
cycle circadien (12). Ils peuvent également présenter des symptômes atypiques tels des troubles
moteurs (1), du langage (1,13) et visuels (13).
2
Il existe deux formes de la AD, soit la familiale (précoce) et la sporadique (tardive). La AD
familiale est observée lors de l’expression génomique de polymorphismes génétiques qui favorisent
l’expression de l’amyloïde ou altèrent le ratio entre les différentes formes d’Aβ, soit l’Aβ1-42, qui
tend à s’agréger, et l’Aβ1-40, qui est la forme plus commune (14,15). Ces variations génétiques ont
été observées au niveau des gènes codant l’APP, la préséniline (PS)-1 et la PS-2. Situés sur le
chromosome 21, 20 polymorphismes du gène APP (Tableau I) ont été identifiés, ce qui correspond
seulement à 5% des cas familiaux. Les cas précoces sont donc principalement causés par des
mutations au chromosome 14 au niveau des gènes PS-1 et PS-2. Quant à ceux-ci, 120 et 8 variations
génomiques ont respectivement été répertoriées (16). Les patients qui les possèdent présentent des
symptômes de la AD pour la première fois vers 40 ans (17). Toutefois, près de 95% des cas de la
AD sont observés dans la population âgée d’au moins 65 ans qui possède la forme tardive de cette
pathologie (18).
À partir des mutations identifiées chez les patients atteints de la forme familiale, des modèles
animaux ont été développés chez la drosophile, le poisson P. marinus, le ver C. elegans et la souris.
Ces modèles permettent de reproduire les aspects généraux de la pathogenèse tels la cascade de
progression, ainsi que les modulateurs et les gènes influencant la AD (16). Les premiers modèles
murins furent développés par l’insertion des gènes humains APP Dutch et APP Flemish. Ces
modèles présentèrent une amyloïdose cérébrale à un âge très avancé, vers 18 mois (14). Par la suite,
Nom Mutation Site de clivage à proximité Caractéristiques pathologiques
Swedish K670N, M671L β-sécrétase
AD, CAA
– A673V APP AD
Flemish A692G
α-sécrétase
AD, Hémorragie cérébrale
– E693G CAA
Dutch E693Q Hémorragie cérébrale
Italian E693K Hémorragie cérébrale
Japenese E693Δ AD
Iowa D694N AD, CAA
– L705V APP CAA
– A713T APP
γ-sécrétase
AD, Hémorragie cérébrale
Austrian T714I AD
French V715M AD
Florida I716V AD
London V717(I/F/G/L) AD, CAA
Tableau I. Formes mutantes du gène APP découvertes dans les cas familiaux. Les mutations du gène
APP sont réparties selon leur position de l’acide aminé muté. Pour chaque mutation, le site de clivage à
proximité de la mutation est indiqué de même que certaines caractéristiques pathologiques générales. AD:
Maladie d’Alzheimer, CAA: Angiopathie amyloïde cérébrale (16).
3
Hsiao et ses collègues développèrent une souris «knock-in» où le gène APP Swedish (APPswe) est
inséré dans le génome par le vecteur de protéine prionique (PrP) du hamster. Cette lignée
transgénique murine fut appelée Tg2576. Contrairement aux modèles précédents, les souris Tg2576
possèdent de nombreuses plaques en plus d’un trouble cognitif dès 9 mois (19). La Tg2576, une
autre lignée transgénique, APP23, exprime le gène APPswe humain, mais sous le promoteur
mThy1.2 (16). Ces derniers modèles développent également des dépôts vasculaires d’amyloïde
caractéristiques de l’angiopathie amyloïde cérébrale (CAA) (14,16) qui est très commune chez les
malades d’Alzheimer (20).
Un autre modèle transgénique intéressant est la lignée double mutante APPswe/PS1 A246E
(APPswe/PS1). Plus précisément, les souris APPswe/PS1 expriment davantage d’Aβ1-42 que les
souris mutantes APPswe et PS1 A246E (21). De plus, dès 3-4 mois, les femelles APPswe/PS1
démontrent des dépôts d’amyloïde (22) alors que, chez les mâles, l’accumulation robuste est
évidente seulement à partir de 6 mois (23). Comparativement aux autres modèles de la AD, la
pathologie se développe plus rapidement chez les APPswe/PS1. Suivant l’accumulation de plaques
séniles, il a été observé que, dans ce modèle, il y a une diminution de l’expression de plusieurs
protéines synaptiques (e.g. AMPA, Arc, Erg1, NR2A/B, PSD95) et de facteurs neurotrophiques
(e.g. BDNF) (23). À un stade plus avancé, le nombre de plaques devient plus important et couvre
près de 80% de l’hippocampe chez certains animaux (21).
1.1.1. Tau
La AD est caractérisée par la formation de neurofibrilles (7,8) localisés dans le corps cellulaire, les
dentrites apicales et distales des neurones, ou encore associés à des plaques amyloïdes dans les
neurites anormaux (24,25). Ce type de lésions neurofibrillaires est également observé dans d’autres
troubles neurodégénératifs tels la maladie de Pick, la dégénération cortico basale, la paralysie
supranucléaire progressive et la démence fronto-temporale (FTD) (24,25). Parmi ces troubles
neurodégénératif, la maladie de Pick et la FTD décrivent la neurodégénération des lobes frontal et
temporal respectivement associé avec une démence progressive présentant des lésions
neurodégénérative riches en tau et des déficits moteurs pouvant contribuer au développement de la
maladie de Parkinson (16). Tout comme la FTD, la dégénération cortico-basale et la paralysie
supranucléaire sont également des troubles neurodégénératifs moteurs entrainant respectivement un
déficit moteur induit par la perte de neurones corticales et extrapyramidales, et une paralysie
occulaire (16)(24,25). Les premières mutations de tau ont été identifiés chez les patients ayant la
FTD (16).
4
La protéine tau semble ainsi participer à la neurodégénération. Plusieurs modèles ont donc été
développés pour étudier son rôle lors de maladies. L’expression de l’isoforme de tau humain la plus
courte de 352 acides aminés (26) ou la plus longue de 441 acides aminés (27), chez des souris
Alzheimer, induit respectivement la formation tardive, vers 18-20 mois (26), ou précoce (27) de
neurofibrilles. Chez les Tg2576, l’intégration de tau muté, tau P301L, augmente massivement le
nombre de neurofibrilles notamment au niveau du cortex entorhinal et l’amygdale, les régions
vulnérables à la déposition d’amyloïde, et ce, sans modifier l’expression de tau (28). En ce sens,
Geula et al ont démontré la capacité de l’Aβ1-42 synthétique à induire la formation de neurofibrilles
chez le singe rhésus âgé (29). En effet, les oligomères d’Aβ sont capables d’indure la
phosphorylation de tau par l’activation de protéines kinases, soit GSK3β, CDK5, MARK et MAPK
(30). Lorsque hyperphosphorylé, tau est clivé à son domaine de liaison aux microtubules ce qui le
dissocie des microtubules. Il devient soluble et est sécrété par les cellules (31).
L’hyperphosphorylation de tau permet également son entrée dans les épines dendritiques où il se
localise anormalement (32). Une fois entré dans les neurones, la conformative native de tau soluble
favorise la formation de filament hélicoïdaux neurotoxiques, puis l’agrégation de ces filaments en
neurofibrilles ce qui contribue au dysfonctionnement synaptique (33,34). Outre l’amyloïde, la
phosphorylation de tau est également induite après un choc thermique, l’hypoxie ou la privation de
glucose (30). Bien que la phosphorylation de tau à des sites supplémentaires soit associée à un
déficit synaptique, le processus d’hyperphosphorylation reste à approfondir (35).
1.1.2. Amyloïde
1.1.2.1. Formation, élimination et dégradation de l’amyloïde
L’Aβ est dérivée de l’APP. Cette glycoprotéine associée à la membrane est transformée par deux
voies dont l’une génère l’amyloïde et l’autre non (Fig. 1.1). Lors de cette dernière, l’APP est clivée
par l’α-sécrétase au niveau de son domaine Aβ sécrétant la portion amino-terminale, le fragment
soluble APP-α (APP-sα), et prévenant la formation d’amyloïde. L’Aβ, quant à elle, provient du
clivage séquentiel de l’APP par la β-sécrétase et le complexe γ-sécrétase (36). La β-sécrétase,
BACE-1, clive l’APP libérant le fragment soluble APP-β (APP-sβ) du fragment carboxy-terminal β
(CTFβ) (36,37). Le CTFβ est alors clivé par le complexe γ-sécrétase qui est composé de la PS, la
nicastrine, protéine du pharynx antérieur défectueux 1 (APH-1) et la protéine activatrice de la
préséniline (PEN-2) (36,38). La longueur de fragment d’Aβ, variant entre 39 et 43 acides aminés
(6), est définie par le clivage via la γ-sécrétase (37). La forme la plus commune obtenue par ce
clivage est du peptide Aβ1-40 (36). L’Aβ est produite directement au cerveau, mais également à la
périphérie. L’Aβ circulante entre au cerveau grâce à l’action du récepteur des produits finaux de
5
glycosylation avancée (RAGE) (Fig. 1.2) (39,40) et ce, uniquement au niveau du système nerveux
central (SNC) (41). En effet, Roberts et ses collègues (2014) détectèrent aucune fluctuation des
concentrations d’amyloïde au niveau des veines périphériques signifiant que l’entrée et la sortie de
l’Aβ s’effectue uniquement au niveau du système nerveux central (SNC) (41).
L’élimination de l’Aβ devient alors un phénomène intrinsèque à l’homéostasie. L’amyloïde est
éliminée du cerveau par divers mécanismes (Fig. 1.2) (42). Notamment, les cellules de l’unité
neurovasculaire (NVU) (Section 1.5 – Unité neurovasculaire) sécrètent des protéases contribuant
à sa dégradation (43), telles que la plasmine, la néprilysine (NEP), la NEP2, l’enzyme de
dégradation de l’insuline (IDE), l’enzyme de conversion de l’angiotensine (ACE), les
métalloprotéases matricielles (MMPs), etc. (44). De plus, certaines cellules de la NVU, astrocytes
(43,45), péricytes (46), et microglies (43,47), peuvent internaliser l’Aβ. Cette internalisation est
généralement dépendante de la protéine 1 apparentée au récepteur des lipoprotéines à faible densité
(LDLs), LRP1. Une fois lié, LRP1 promeut l’internalisation de son ligand et le dirige vers les
lysosomes où il sera dégradé (48,49). De plus, LRP1 et la p-glycoprotéine (PgP) contribuent à la
transcytose rapide de l’amyloïde soluble, de l’Aβ associée à l’α-macroglobuline (42,50) et de l’Aβ
associée à l’apolipoprotéine (Apo) E2 ou 3 (51) à travers la barrière hémato-encéphalique (BBB) au
Figure 1.1. Voies de transformation de l’APP. L’APP via deux cascades, l’une non-amyloïdogénique et
l’autre amyloïdogénique. L’APP est clivée par l’α-sécrétase formant deux fragments, APP-sα et CTFα.
l’APP peut également être endocyter, puis clivé par la β-sécrétase produisant le fragment APP-sβ et le
CTFβ. Celui-ci forme alors l’Aβ lorsque clivé par la γ-sécrétase. APP-sα: L’amyloïde est ensuite sécrétée
dans le milieu extracellulaire où son accumulation promeut la formation de plaques (44) [tiré
intégralement].
6
sang veineux (48,49) et à travers le plexus choroïde au fluide cérébrospinal qui est ensuite
réabsorbée par les veines cérébrales (52). Une équipe en particulier , permirent entre-autre d’évaluer
l’importance de l’élimination de l’Aβ du CNS à travers la BBB à 25% et celle via les plexus
choroïdes également à 25% (41). En plus d’exprimer LRP1 (53,54), les cellules de la BBB
expriment ABCB1 (50,55) et LRP2 (56) modulant le transport de l’Aβ. Plus précisément, LRP2
orchestre l’influx et l’efflux du complexe formé par l’Aβ et la clusterine, également appelée ApoJ
(56). Tout comme LRP1, LRP2 est impliqué au niveau de la dégradation de l’Aβ par endocytose, de
même que son élimination par la BBB lorsque celle-ci est associée à l’ApoJ (57). L’Aβ peut former
un complexe avec l’ApoE. Celle-ci possède trois isoformes: ApoE2, ApoE3 et ApoE4 (58), dont
chacun module différemment l’élimination de l’Aβ. Comparativement à l’ApoE2 et l’ApoE3, le
complexe de l’Aβ avec l’ApoE4 est internalisé via le récepteur LRP1 et éliminé du cerveau
beaucoup plus lentement (51,59,60). Ainsi, l’ApoE4 a un effet de rétention de l’Aβ au cerveau.
Suite au passage à travers la BBB, l’Aβ se lie à LRP soluble (sLRP) qui est produit par le clivage de
LRP par la β-sécrétase (61). LRPs agit alors comme un «siphon périphérique» captant l’Aβ du
cerveau dans le sang et la transportant jusqu’au foie (62) où elle sera dégradée (62,63).
Figure 1.2. Voie d’entrée et d’élmination de la β-amyloïde. Les voies d’entrées et d’élimination de l’Aβ
démontre l’importance de l’unité neurovasculaire à son homéostasie. L’Aβ est produite à partir de l’APP
au cerveau et à la périphérie. L’Aβ est dégradée enzymatiquement par la néprylisine, l’IDE, les
métalloprotéinase et la plasmine. L’Aβ non-dégradée peut s’oligomériser et être dégradée par les
microglies et les astrocytes. L’efflux de l’Aβ est médiée par LRP1/2 alors que l’influx est modulé par
RAGE. L’Aβ s’associe à LRP1 soluble dans la circulation sanguine. L’élimination systémique de l’Aβ se
produit au niveau du foie. CLU: Clusterine. (42) [tiré intégralement].
7
1.1.2.2. Agrégation de l’amyloïde
Toutefois, avec l’âge ou en condition pathologique, ces voies d’élimination de l’Aβ sont altérées
(64). Par exemple, l’activité enzymatique de l’IDE et de la NEP diminuent avec l’âge (65). De plus,
l’activité de la NEP2 (66) et l’expression de la NEP (67) diminuent chez les patients déments. Chez
les malades d’Alzheimer, il a également été observé que LRPs est oxydé (62) en plus d’une
expression accrue de RAGE à la surface des cellules endothéliales de la BBB (53). Ces deux
phénomènes contribuent respectivement à la réduction de la liaison de l’Aβ à LRPs oxydé (62) et
l’augmentation de l’entrée d’Aβ au cerveau (53). Avec l’âge et lors de la AD, la capacité des
microglies à dégrader l’Aβ décroît (68). L’ensemble de ces altérations contribue à l’accumulation
de l’Aβ et la formation d’agrégats (53). Grâce à une étude de spectrométrie de masse, la dynamique
de nucléation des plaques amyloïdes a pu être décrite. Bernstein et ses collègues ont déterminés que
l’Aβ1-40 formait un dimère puis un tétramère, alors que l’Aβ1-42 formait des dimères, tétramères,
hexamères, puis dodécamères. Cette structure de dodécamère constitue le noyau des agrégats
pouvant former, par un processus lent, des protofibrilles (69). Les plaques amyloïdes sont formées
principalement de ce nucléus, en plus d’autres protéines dont l’α1-antichymotrypsine (70), l’ApoE
(71), le protéoglycane à héparane sulfate (71) et la thrombospondine (72). Avec le temps, ces
plaques amyloïdes grossissent et s’organisent en feuillet-β jusqu’à se stabiliser tel que confirmé par
leur suivi longitudinal in vivo (73).
1.1.2.3. Hypothèse de la cascade amyloïde
Outre la AD, la déposition d’amyloïde est commune à plusieurs troubles neurodégénératifs tels le
syndrome de Down (74), la maladie de Parkinson, la maladie d’Huntington (75), la démence
vasculaire, la CAA et l’atrophie corticale postérieure (76), ce qui suggère une étroite relation entre
la neurodégénérescence et l’amyloïde. Cette théorie est d’ailleurs renforcie par plusieurs évidences
génétiques sur le gène APP identifiées chez des malades Alzheimer et dont, lorsqu’exprimé chez
des modèles animales, suffisent au développement de la AD (16). De ce fait, les scientifiques ont
émis l’hypothèse de la cascade amyloïde. Elle postule que l’Aβ initie une cascade cellulaire
provoquant la perte neuronale et des dommages neuronaux qui ont initialement été attribués au
nombre de plaques séniles (36). Toutefois, le déclin cognitif ne corrèle pas avec les dépôts
d’amyloïde et la perte synaptique (77). Dès lors, l’hypothèse de la cascade amyloïde a été révisée
(36,78). Il a alors été proposé que l’Aβ soluble, oligomère et dodécamère (69), serait à l’origine de
la neurotoxicité (79,80). Les oligomères d’Aβ induisent l’hyperphosphorylation de tau (Section
1.1.1 – Tau) (29,30). Certes, ils peuvent également interagir avec plusieurs protéines neuronales
dont: la neuroligine, les récepteurs nicotiniques-α7, les récepteurs adrénergiques, les canaux
8
calciques (81), les récepteurs ionotropiques AMPA (82), les récepteurs ionotropiques NMDA (83)
et les récepteurs métabotropiques du glutamate 5 (mGluR5) (84). En se liant aux neurones,
l’amyloïde induit une signalisation aberrante (e.g .voie Wtn) un influx calcique anormal (81), en
plus d’une activité et d’une plasticité synaptique altérées (81,85). Dans leur ensemble, les effets
induits par les oligomères d’Aβ contribuent au déclin cognitif.
L’Aβ module également l’expression de gènes critiques à l’apprentissage, la mémoire et la
neuroprotection (86). Par exemple, les fibrilles amyloïdes inhibent l’expression de la neuroligine, ce
qui promeut la neurodégénération (87). Mis à part les oligomères et les fibrilles, l’amyloïde
intracellulaire contribue également à la toxicité au sein de la mitochondrie résultant en un
dysfonctionnement mitochondrial reconnu chez les malades d’Alzheimer (88). L’implication des
fibrilles, des oligomères insolubles et de l’amyloïde intracellulaire reste à explorer afin de préciser
cette hypothèse.
Figure 1.3. Schéma de l’hypothèse vasculaire de la maladie d’Alzheimer. Les facteurs vasculaires
provoquent le premier dommage: la dysfonction de la BBB et l’oligémie. La dysfonction de la BBB
provoque une accumulation de substances toxiques et une diminution de l’élimination de l’Aβ. L’oligémie
entraîne une hypoperfusion capillaire en plus d’une augmentation de l’expression et de la transformation
de l’APP. Cela provoque le deuxième dommage : l’augmentation du niveau d’Aβ. L’augmentation de
l’Aβ entraîne une hyperphosphorylation de tau et, favorise le dysfonctionnement neuronal conduisant à
long terme à la démence (42) [tiré intégralement].
9
1.1.3. Hypothèse vasculaire
Récemment, une autre hypothèse, considérant les facteurs de risque de la AD, a vu le jour, soit
l’hypothèse vasculaire (Fig. 1.3). Elle suppose qu’un dommage initial de la microcirculation
cérébrale initie le dysfonctionnement neuronal non-amyloidogénique. Cette voie non-
amyloidogénique comporte une rupture de la BBB, induisant une perte d’étanchéité et la sécrétion
de molécules neurotoxiques. De plus, le dommage vasculaire occasionne une oligémie pouvant
provoquer des ischémies focales. Ces deux phénomènes provoquent une augmentation de l’Aβ
respectivement par la perte de l’élimination et par une élévation de la production de l’Aβ (42). En
effet, des études ont démontré qu’un contexte hypoxique ou ischémique favorisait la conversion de
l’APP en Aβ en induisant l’augmentation de l’activité de la β-sécrétase et de la γ-sécrétase, en plus
de l’expression de la β-sécrétase (89). Loin de contredire l’hypothèse de la cascade amyloïde,
l’hypothèse vasculaire considère la dynamique de la pathogenèse de la AD. Cependant, le processus
pathologique lui-même est encore peu connu. L’étude des comorbidités telles que les troubles
vasculaires permettent de dévoiler la cascade pathologique de la AD.
1.2. Facteurs de risque de la maladie d’Alzheimer
La AD possède une étiologie complexe qui progresse sur une vie. L’âge est le facteur ayant le plus
d’incidence sur la progression de la forme sporadique (90). De plus, la génétique (91,92), l’obésité,
le diabète (93,94), l’accident vasculaire cérébral (AVC) (93–96), l’hypertension (93–95),
l’hypotension (95), les maladies coronariennes (95,96), la fibrillation auriculaire (97),
l’athérosclérose (93,98) et l’hypercholestérolémie (93) représentent des facteurs de risque de la AD
tardive. Nous résumerons brièvement l’implication de la génétique, soit l’incidence des différents
isoformes d’ApoE, de l’obésité, du diabète et des troubles vasculaires dans la pathogenèse de la
AD.
1.2.1. Génétiques
ApoE4 constitue le facteur génétique principal de la AD tardive. Les personnes porteuses de l’allèle
ApoE4 présentent 4 à 10 fois plus de risque de développer la AD (91,92). Chez l’homme, le gène
ApoE se localise sur le chromosome 19 (92) pour lequel 3 allèles du même locus génétique ont été
identifiées, ApoE2, ApoE3 et ApoE4 (58,92). L’isoforme ApoE4 est la plus primitive (99) et la
moins prévalente (100). Au sein de la population, 30%, 60% et 10% de la population possède
respectivement l’isoforme ApoE2, ApoE3 ou ApoE4 (100). Ces isoformes varient d’une
substitution d’un seul acide aminé au niveau de deux résidus: ApoE2 (Cys112, Cys158), ApoE3
10
(Cys112, Arg158) et ApoE4 (Arg112, Arg158) (101). Ces mutations ont des effets majeurs sur sa
structure et sa fonction (101).
L’ApoE est produite principalement par les astrocytes au cerveau (102) et par le foie à la périphérie.
Circulant dans le sang, elle est intégrée aux lipoprotéines de très faible densité et aux chylomicrons.
L’ApoE régule le transport du cholestérol et des lipides. Elle module également l’élimination des
lipoprotéines plasmiques par les récepteurs LDL (103). Tel qu’évoqué plus tôt, l’ApoE module le
transport de l’Aβ selon l’isoforme exprimée. L’ApoE2 et l’ApoE3 facilitent l’élimination de l’Aβ,
alors que l’ApoE4 favorise sa rétention (51). L’ApoE2 possède une structure plus stable due à la
substitution d’acides aminés, ce qui lui confère un effet protecteur contre la AD (101). L’ApoE2 et
l’ApoE3 contribuent également à la plasticité synaptique et la réparation neuronale (100). À
l’opposé, l’ApoE4 est étroitement associée au dysfonction de la BBB (104), à l’augmentation de
l’incidence des maladies vasculaires (105), de la CAA (106) et celle de la AD (91,92).
1.2.2. Obésité
Chez la personne obèse, les tissus adipeux, plus importants, produisent des cytokines qui seront
sécrétées dans la circulation sanguine (e.g. TNFα, IL-6). Ces cytokines circulantes peuvent altérer la
fonction endothéliale et contribuer à la résistance à l’insuline (107). En cours de vie, ce phénomène
résulte en une augmentation du risque de diabète de type 2 (108) et des maladies cardiovasculaires
dont l’hypertension artérielle (109). Ces maladies secondaires à l’obésité sont reconnues comme
ayant des effets nocifs sur le cerveau (94). De ce fait, il a été reporté que l’obésité à mi-vie est
associée à un déclin cognitif en fin de vie (93,94,110).
1.2.3. Diabète
Le diabète favorise le déclin cognitif, ce qui promeut la AD (93,94). Sonnen et ses collaborateurs
ont reporté une aggravation de la démence chez les malades d’Alzheimer diabétiques non contrôlés
par rapport à ceux qui sont traités (111). De ce fait, lors d’épisodes d’hyperinsulinémie, le niveau
d’insuline cérébrale augmente. L’insuline cérébrale compétionne alors avec l’Aβ extracellulaire en
tant que ligand de l’IDE, ainsi la dégradation de l’amyloïde par l’IDE est réduite (112).
L’hyperinsulinémie entraîne également une altération de la signalisation de l’insuline, du stress
oxydatif et des mécanismes inflammatoires pouvant notamment contribuer au déclin cognitif (113).
De plus, le diabète peut entraîner des complications dont les maladies vasculaires, les
néphropathies, les neuropathies et les rétinopathies (114). L’augmentation de l’incidence de
démence chez les diabétiques a été d’ailleurs attribuée aux maladies cardiovasculaires (115).
11
1.2.4. Troubles vasculaires
En plus d’être des facteurs de risque de la AD, l’expression d’ApoE4, l’obésité et le diabète
accroient les risques de développer des troubles vasculaires (105,109,115). Plusieurs études
épidémiologiques, cliniques et animales ont démontré que les troubles vasculaires systémiques tout
comme les maladies cardiovasculaires en mi-vie contribuent à la défaillance cognitive (93,116).
Nous comptons parmi ces pathologies: l’AVC (93–96), l’athérosclérose (93,98), la fibrillation
artriale (97), l’hypercholestérolémie (93), l’hypertension (93–95,117,118), l’hypotension
(95,117,118) et les maladies coronariennes (95,96).
1.3. Hypoperfusion
Afin de bien fonctionner, le cerveau nécessite un approvisionnement constant et régulé de
nutriments et d’oxygène lequel est orchestré par le CBF. En fait, le cerveau est un des organes les
plus actif et consomme jusqu’à ~20% de l’oxygène et ~25% du glucose consommés par le corps ce
qui correspond à 20% du débit cardiaque (119). Qui plus ait, depuis la dernière décennie,
l’importance de la perfusion sanguine cérébrale a été à de nombreuses reprises démontrée et ce, par
les effets délétères qu’à une réduction du CBF. La cas échéant, nous observons une perturbation des
effecteurs de la mémoire (120) conduisant à une altération de l’apprentissage et de la mémoire
(121).
1.3.1. Régulation du flux sanguine et maladie d’Alzheimer
En condition physiologique, la NVU effectue des ajustements vasculaires afin de maintenir le CBF
stable. Elle régule égalment la distribution du sang selon les demandes énergétiques (121). Cette
régulation du CBF est appelée couplage neurovasculaire (119,121). En cas de troubles vasculaires,
des événements cellulaires et moléculaires sont déclenchés qui entraînent un dysfonctionnement de
la NVU (95) suivi d’une réduction du flux cérébral sanguin (CBF) (95,96,98,117,122). La NVU est
alors incapable de combler les demandes énergétiques des régions actives du cerveau (121) et de
contrôler le CBF (95). Cette perte de l’autorégulation s’observe par des fluctuations du flux saguin
chez les souris transgéniques Alzheimer (123), les malades d’Alzheimer (118,124) et ceux MCI
(124,125). Suite à ces évidences, plusieurs ont tenté de démontrer la relation entre l’hypoperfusion
et la démence. Une étude intéressante à grande échelle a permis de confirmer cette hypothèse (126).
D’autres études épidémiologiques suggèrent une contribution de l’hypoperfusion dans la
pathogenèse de la AD (127,128). Par la technique de tomographie à émission de positrons (PET),
Hunt et ses collègues ont également démontré une réduction du métabolisme du glucose cérébral
chez des individus MCI situant l’altération vasculaire antérieure à la démence (129). Selon
12
l’hypoperfusion cérébrale, il a été observé une crise énergétique (95,130) où l’apport en glucose et
en oxygène est réduit aux régions vulnérables (131). La déficience en glucose, causée par
l’hypoperfusion, induit un stress oxydatif et réticulaire aux neurones hippocampaux et corticaux
restreignant leur production d’ATP (132). Ainsi, l’hypoperfusion induit un dysfonctionnement
neuronal (133) ce qui altère l’intégrité et la structure du cerveau (93,134) et, contribue à la
neurodégénérescence et au déclin cognitif (95,130) ; phénomènes qui aggravent la AD (42).
L’hypoperfusion induit également l’altération de l’élimination et/ou du transport de l’Aβ (93,121).
Par conséquent, l’hypoperfusion induite par les troubles vasculaires initierait ou accélèrerait la
cascade pathologique de la AD (42,135).
1.3.2. Oligémie versus ischémie
Il existe deux formes d’hypoperfusion cérébrale: l’oligémie et l’ischémie. L’oligémie décrit un
processus lent pouvant prendre des mois ou des années à s’intaller, alors que l’ischémie réfère à une
réduction assez rapide et soudaine du flux sanguin entraînant la mort de cellules au niveau d’une
lésion dite ischémique (136). Suite à l’oligémie induite par la ligature unilatérale de l’artère carotide
commune (1VO), le déclin cognitif est exacerbé chez les Tg2576 (137) et les APPswe/PS1 (138).
Le même effet synergique a été observé chez les J20/APP (139) soumises à une ischémie induite
par la sténose bilatérale des artères carotides communes (BCAS).
L’oligémie induit une réduction modérée du CBF associée à une diminution de la synthèse
protéique (121,140). Certaines anomalies de la NVU ont également été observées suite à l’oligémie,
dont l’altération de l’interaction intermodale axone-glie (141), l’épaississement de la membrane
basale et la déposition de collagène sur la BBB (142). L’altération de la capacité mnésique décrite
est causée par un dysfonctionnement neuronal (138,140), puisqu’aucune mort cellulaire n’est
observée. Quant à l’ischémie, la réduction du CBF est plus importante provoquant généralement
une hypoxie (42). L’hypoxie entraîne l’expression du facteur de transcription, HIF-1α, qui
augmente l’expression de BACE-1 (89) et diminue l’expression de la neprilysine (143). De ce fait,
les souris Tg swe/dutch/iowa soumises à la BCAS développent plus rapidement des dépôts
amyloïdes associés à un stress ischémique (144). Lorsque la réduction du CBF est supérieure à
50%, celle-ci promeut un dysfonctionnement de la synthèse de l’ATP et une altération de l’activité
neuronale (119). De surcroît, l’ischémie altère l’expression des protéines neurotrophiques et
neuronales (145–147). Suite à l’ischémie, l’expression de MMP-2 augmente (148) alors que
l’expression de claudine V et occludine diminuent (149), occasionnant une altération de la BBB. La
rupture de la BBB interrompt le transport membranaire normal des nutriments, des vitamines et des
13
électrolytes, nuisant encore une fois au fonctionnement neuronal (150). Contrairement à l’oligémie,
plusieurs études, basées sur le modèle de l’occlusion bilatérale des artères carotides communes
(BCAO) ou de la BCAS, ont démontré que les dommages de la matière blanche et la perte
neuronale sont à l’origine du déclin cognitif (139,149,151–157). Ces évidences indiquent un rôle
central de l’hypoperfusion cérébrale, l’oligémie et l’ischémie, dans la pathogenèse de la AD. Bien
que davantage d’études portent sur l’ischémie, la relation entre la AD et l’oligémie représente un
sujet à approfondir, puisque les changements du CBF observés avec le vieillissement ressemble
davantage à l’oligémie (130,142).
1.4. Système de l’activateur tissulaire du plasminogène
Suite à un stress vasculaire, tel que la formation d’un caillot sanguin ou thrombus occludant un
vaisseau sanguin, l’activateur tissulaire du plasminogène (t-PA), une sérine protéase, est produit et
libéré par les cellules endothéliales afin de lyser le caillot (158). Il s’ensuit la conversion par clivage
protéolytique du plasminogène en plasmine par le t-PA (159–161). La plasmine contribue alors à
dégrader le thrombus en dégradant la fibrine (162) et la laminine (163). Ce système du
plasminogène est régulé à plusieurs niveaux. L’inhibiteur 1 des activateurs du plasminogène (PAI-
1) inhibe le t-PA (159,160,164), alors que la neuroserpine interagit uniquement avec le t-PA
(159,160,165,166). Quant à la plasmine, elle est inhibée par l’α2-anti-plasmine et l’α2-
macroglobuline (159,160). Le complexe formé avec les protéines inhibitrices est généralement
internalisé par les cellules via LRP1, puis dégradé (167). Dans la circulation, le t-PA possède une
demi-vie très courte, d’environ 5 minutes (168,169), suivant laquelle il est éliminé de la circulation
par LRP1 au niveau du foie pour y être dégradé (170). Jusqu’à ce jour, une forme recombinante du
t-PA (rt-PA) représente le seul traitement pour l’AVC ischémique (171), l’un des facteurs de risque
de la AD (93–96).
1.4.1. Fonction du système de l’activateur tissulaire du plasminogène
Présent endogéniquement dans la circulation comme enzyme thrombolytique (172), le t-PA est
également exprimé au SNC par les astrocytes, les microglies et les neurones, soit à l’amygdale
(173), au cervelet (174), au corps calleux (175), à l’hippocampe (165,173,174,176,177), à
l’hypothalamus (173,176), etc. Au niveau du SNC, la majorité du t-PA qui agit sur celui-ci est
produit endogéniquement et ce, par les cellules endothéliales de la BBB (158). Or, le t-PA circulant
et exogène au cerveau peut également agir au niveau de celui-ci directement en traversant la BBB
par transcytose médié par LRP1 (178). Plus précisément, le t-PA est une glycoprotéine de 69 kDa
formée d’une seule chaîne de polypeptide organisé en 5 domaines: Kringle 1, Kringle 2, epidermal
14
growth factor(EGF)-like, en doigt et protéase (Figure 1.5) (179,180). Ce sont d’ailleurs ces
différents domaines qui lui confèrent son effet pléiotropique au sein du SNC. Le t-PA possède une
activité thrombolytique (162,163). Son activité enzymatique lui confère également des propriétés
neuroprotectrices. En effet, au SNC, cette sérine protéase contribue notamment à moduler la
plasticité synaptique (181–186), la potentialisation à long-terme (LTP) (170,173,187,188) et la
dépression à long-terme (LTD), mais également la perméabilité de la BBB (170,179,189,190) et la
réponse inflammatoire du cerveau (191–197).
Grâce à son activité enzymatique, le t-PA convertit des pro-neurotrophines en leur forme active
(e.g. BDNF, NGF) (198,199) et participe au remodelage neuronal par la production de vésicules
contenant la synaptophysine (183). Par la digestion des protéines de la matrice extracellulaire, le t-
PA module la pousse axonale et, donc, la plasticité synaptique (185). Certes, certains des effets
neurotrophiques du t-PA sont plutôt orchestrés par son action de cytokine qui est elle-même médiée
Figure 1.4. Structure et fonction de l’activateur tissulaire du plasminogène (t-PA). Le t-PA est composé
de 5 domaines: Kringle 1, Kringle 2, facteur de croissance, doigt de zinc et protéase. Le domaine Kringle 2
interagit avec l’unité NR1 du récepteur NMDA. Le domaine de facteur de croissance peut se lier au
récepteur d’EGF et le récepteur du mannose-6-phosphate. Le domaine Kringle 2 et le domaine en doigt
possèdent plusieurs ligands, soit la fibrine, le plasminogène, l’annexine II et les antagonistes du t-PA (PAI-1
et neuroserpine). Le domaine en doigt serait également le domaine liant LRP et potentiellement le domaine
du facteur de croissance. PDGF-CC: Facteur de croissance CC dérivé des plaquettes. (174) [tiré
intégralement].
15
par LRP1 (200). Le t-PA promeut également l’élongation des neurites par l’induction de kinases
trophiques tel que ERK, la protéine kinase C et PI3K/Akt (201). La modulation de voie de
signalisation par le t-PA lui confère entre-autre un effet anti-apoptotique sur les oligodendrocytes
(175) et module l’apoptose neuronale (202,203). Toutefois, une activité excessive du t-PA a été
associée à l’induction de la mort neuronale (204). Kim et ses collègues ont démontré que le t-PA
empêche la mort par stress oxydatif des neurones par son action de cytokine, mais n’interrompt pas
l’apoptose et l’excitotoxicité de ceux-ci (205). D’autres travaux proposent plutôt que le t-PA même
induit l’excitotoxicité (163,175,206). Celle-ci peut être induite par le clivage du récepteur NMDA
(175,206) au niveau de sa sous-unité NR1 à laquelle le t-PA se lie (207). En plus, le t-PA se lie à
LRP (208) ce qui induit des signaux intracellulaires et promeut le flux calcique altèrant transmission
(209,210). Ces dernières actions par le t-PA contribue alors à la LTD (211).
De nombreuses études mettent en évidence la modulation de la perméabilité de la BBB par le t-PA
circulant (170,179,189,190). En se liant à LRP1 (170,212), le t-PA déclenche des signaux
intracellulaires menant également à l’activation de MMP2/9 (212–215). Le t-PA module la
perméabilité de la BBB suite à un stimulus (e.g. ischémie) qui, lorsque soutenu, s’avère nocif et
peut provoquer la rupture de la BBB (216,217) et l’extravasation de fluide dans l’espace
périvasculaire causant un œdème (217). En fait, lors de l’administration à l’extérieur de la fenêtre
d’intervention thérapeutique, 4,5 heures suivant l’AVC (218), ou la sur-administration (219) du rt-
PA suite à un AVC se produit, davantage d’effets néfastes que bénéfiques sont observés. En effet,
l’administration tardive et le sur-dosage du t-PA augmentent les risques d’hémorrhagies cérébrales
(219–221).
Par sa liaison avec LRP1 (222) ou l’annexine II (195), le t-PA est également apte à moduler
l’inflammation en induisant le recrutement des macrophages (196), l’activité microgliale (192–195),
en plus de la production de cytokines pro-inflammatoire (191–194). Une étude avait également
reporté un rôle de cytokine anti-inflammatoire au t-PA (197). Cette activation des microglies
s’avère nécessaire à la neurodégénération excitotoxique des neurones hippocampaux
quoiqu’insuffisante à l’initier (177). Par conséquent, le t-PA possède plusieurs rôles et
dépendemment du contexte son activité devient bénéfique ou néfaste pour le cerveau.
1.4.2. Système de l’activateur du plasminogène et maladie d’Alzheimer
Dans les modèles transgéniques murins, le t-PA est fortement exprimé autour des plaques denses
amyloïdes résultant en leur arrêt de croissance ou leur dégradation (162,223,224). Les agrégats
d’Aβ de type fibrilles stimulent l’expression du t-PA (225) ce qui entraîne la dégradation de
16
l’amyloïde via la plasmine (225,226). Le t-PA contribue également à la dégradation de l’Aβ
indirectement par l’induction de l’activation de MMP2/9 (213–215). De plus, l’Aβ régule
positivement la neuroserpine (227) qui intéragit directement avec l’Aβ et limite la formation de
fibrilles (228). Des études avec des souris «knock-out» du PAI-1 (229) et de la neuroserpine (230)
révèlent une réduction de la quantité d’amyloïde soulignant l’importance du t-PA dans l’élimination
de l’Aβ. Cet effet du t-PA a également été observé suite à l’inhibition pharmacologique de
régulateur de la plasmine (231). Dans cet ordre d’idée, la déplétion du t-PA augmente le nombre de
plaques en plus d’aggraver les déficits cognitifs chez les Tg2576/t-PA+/– (232). Malheureusement,
avec le temps, le système du plasminogène devient inefficace à dégrader des dépôts d’amyloïde
(162). En effet, le t-PA réduit dramatiquement avec l’âge et la AD (162,175,198,233,234).
Quoiqu’une certaine étude n’a pas observé d’altération du système du t-PA (235), la majorité des
travaux effectués chez les patients et les souris transgéniques démontrent une diminution de
l’expression et de l’activité du t-PA (162,227,233,234) et de la plasmine (226,227), alors que celle
du PAI-1 (162,236) et de la neuroserpine (227) augmentent. Dans ces conditions, la déposition de
fibrine augmente (237,238). Il s’en suit alors des dommages neurovasculaires, en plus d’une
réaction inflammatoire (239). La dysfonction du t-PA entraîne également des déficits sévères de la
plasticité synaptique (211) ce qui contribue à la progression de la AD. Ainsi, le t-PA et les protéines
du système du plasminogène représentent des cibles thérapeutiques potentielles.
1.5. Unité neurovasculaire
Tel que brièvement mentionné plutôt, la NVU (Fig. 1.5) représente l’unité intrinsèque au sein de
laquelle les cellules de la BBB (cellules endothéliales et péricytes), les cellules gliales (astrocytes,
oligodendrocytes et microglies) et les neurones interagissent et communiquent étroitement entre eux
(119,121). Les cellules endothéliales composent l’endothélium des microvaisseaux où elles forment
des jonctions serrées et adhérentes qui limitent les échanges passifs (119,240). Les péricytes
entourent près de 80% de la surface de ces microvaisseaux (241) via un contact peg-socket essentiel
à leur maintien, qui constitue des projections cytoplasmiques entrelancées ancrant les péricytes sur
les cellules endothéliales (242). Les pieds astrocytaires entourent les cellules endothéliales
complétant la BBB et permettant la communication entre les neurones et les cellules de la BBB
(243), en plus de maintenir la fonction de la celle-ci (244). L’intégrité de cette unité fonctionnelle
est essentielle au bon fonctionnement du cerveau (121). La NVU contribue notamment à la
maintenance de l’homéostasie cérébrale, au couplage neurovasculaire (119,121,243,245,246), à la
perméabilité de la BBB (119,240,247), etc.
17
1.5.1. Fonction de l’unité neurovasculaire
Par exemple, la BBB forme une barrière physique et sélective (247) régulant les échanges de
métabolites et de nutriments nécessaires au bon fonctionnement neuronal (119). La BBB est donc
cruciale à la maintenance de la composition du fluide interstitiel du cerveau, mais également à
l’élimination de macromolécules potentiellement toxiques (119) telles l’Aβ (68).
Les péricytes, quant à eux, sont des cellules contractiles modulant le CBF via la constriction de la
paroi des vaisseaux sanguins (246). Ils expriment d’ailleurs plusieurs protéines associées à la
contraction dont l’α-actine spécifique aux muscles lisses, la tropomyosine et la desmine (248). Ils
stabilisent également les capillaires nouvellement formés (249). En effet, ils jouent un rôle
important dans la régulation de la prolifération, la survie et la migration des cellules endothéliales,
en plus de moduler les connections des vaisseaux cérébraux (250). Ils prennent part à
l’inflammation du fait de l’expression de récepteur de l’immunité innée (251) et leur capacité de
recrutement de leucocytes au site d’inflammation (252,253). Qui plus est, les péricytes possèdent
une activité semblable aux macrophages induite par la signalisation de récepteurs tels le récepteur
de type Toll(TLR)-4 (254,255) et LRP1 (46). Cela leur permet d’ailleurs d’internaliser l’Aβ pour le
dégrader (46).
Figure 1.5. Représentation de l’unité neurovasculaire et de la microcirculation cérébrale. Les artères
piales, situées dans l’espace sous-arachnoïdie, se divisent en capillaires innervant le parenchyme. Les
cellules endothéliales entourées par les péricytes et en contact avec les pieds astrocytaires forment une
barrière étanche, la BBB. La BBB interagie avec les neurones et les microglies ce qui forme une unité
fonctionnelle, la NVU. SAS: Espace subarachnoïde, VSMC: Cellules vasculaires des muscles lisses. (42)
[tiré intégralement].
18
Comme les péricytes, les astrocytes influencent le CBF (243,245) et aident à l’organisation des
nouveaux capillaires (256). Ils contribuent également à la réponse immunitaire par leur capacité à
internaliser des macromolécules neurotoxiques (43,45) et à recruter des cellules
immunocompétentes (253,257). De plus, ils participent au guidage neuronal, lors du
développement, en agissant telle une cellule d’échafaudage (258). Comme les neurones, les
astrocytes ont besoin pour fonctionner d’un apport continu en oxygène et en nutriments, ce qui
dépend de l’intégrité fonctionnelle de la NVU (259). Lors d’une demande énergétique par les
neurones, les astrocytes servent d’intermédiaires à la communication entre les neurones et les
cellules endothéliales (260).
Les microglies, quant à elles, sont les cellules immunes principales du SNC (261).
L’autoréplication, la division de progéniteurs du cerveau (261) ou l’infiltration de monocytes
circulants précurseurs, Ly6CHigh/CCR2+ (262), permettent de maintenir la population de microglies
constante. Elles constituent l’une des principales lignes de défense contre les pathogènes infiltrant le
cerveau. En condition physiologique, les microglies sont quiescentes et sondent dynamiquement
l’environnement pour détecter toutes molécules toxiques (263). Lorsque la microglie détecte une
menace potentielle, celle-ci s’active vers un profil inflammatoire, M1, ou alternatif, M2 (264). Par
exemple, lorsque la microglie détecte la présence d’Aβ, elle adopte un profil M1 et est recrutée au
site d’agrégation de l’amyloïde (265,266) où elle la phagocytera (43,47). Suite à son recrutement,
l’Aβ promeut également la sécrétion de cytokines et chimiokines pro-inflammatoires par la
microglie (267). Par la sécrétion de chimiokines et de cytokines, les microglies recrutent les
monocytes (253) induisant la réponse inflammatoire. À l’inverse, la microglie activée M2 est
associée à une fonction de maintenance ou de réparation tissulaire. Trois sous-types de la M2 ont
été décrits, nommés M2a, M2b et M2c. Ces sous-types remplissent des rôles distincts, soit la
réparation tissulaire (M2a), l’immunorégulation (M2b) et l’état endocytique (M2c) par lequel la
microglie élimine efficacement les débris et contribue à la réparation, malgré sa faible présentation
d’antigènes (268). La réponse immunitaire contribue à la maintenance et à la régénération axonale
(269). L’ensemble de ses fonctions est d’ailleurs modulé suivant un stress. Notons, toutefois, que
cette classification dogmatique est utilisée afin de simplifier les mécanismes complexes d’activation
des cellules microgliales. En effet, il n’existe pas deux immunophénotypes précis, mais plutôt un
ensemble de différents états de polarisation pouvant être plus ou moins extrêmes.
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1.5.2. Remodelage de l’unité neurovasculaire et maladie d’Alzheimer
Ainsi, la communication dynamique et le fonctionnement de l’ensemble des cellules de la NVU
sont nécessaires au bon fonctionnement du cerveau (119,121). Toutefois, en condition
pathologique, l’unité devient dysfonctionnelle ce qui a des répercutions détrimentaires sur le
fonctionnement du cerveau, soit la fonction cognitive.
Avec l’âge, des changements se produisent au sein de la NVU: diminution de l’expression de
protéines de jonctions serrées, perte de péricytes (242), défaillance du couplage neurovasculaire
(270), réduction du CBF (135) et diminution de la capacité phagocytique des microglies (68). La
perte de péricytes induit une dysfonction des microvaisseaux ce qui a été proposé comme
événement initiant la neurodégénérescence (Section 1.1.3 – Hypothèse vasculaire) (271). Cette
déficience de péricytes augmente la perméabilité de la BBB permettant l’entrée de molécules
toxiques (241). Ce phénomène est également observé suite à des troubles vasculaires tels que
l’AVC ischémique (179,272) et la AD. Chez les malades d’Alzheimer, ces altérations de la NVU
sont plus importantes résultant alors en une accumulation de l’amyloïde (93,119). L’élévation de
l’Aβ cérébrale est d’autant plus importante due à la réduction d’activité des protéines de
dégradation, dont la plasmine (Section 1.4 – Système de l’activateur du plasminogène) (227,230).
Avec la progression de la maladie, les niveaux de chimiokines et cytokines pro-inflammatoires, les
molécules d’adhésion et d’autres médiateurs inflammatoires augmentent considérablement (273).
Par conséquent, le cerveau devient un environnement inflammatoire ce qui pourrait permettre
d’éliminer les macromolécules neurotoxiques du cerveau. Cependant, les macrophages deviennent
avec l’âge moins efficace à éliminer l’Aβ (274), en plus d’avoir une présentation réduite des TLRs
(275). Une inflammation soutenue et une réduction de l’élimination de l’Aβ se produient alors et
contribuent à la neurodégénérescence (68). De plus, il a également été identifié chez des malades
d’Alzheimer qu’une diminution de l’expression du transporteur du glucose, GLUT1, survient
occasionnant une diminution de l’apport de nutriments et substrats essentiels (276). Ce phénomène
est d’ailleurs présent chez des individus asymptotiques susceptibles de développer la AD tel que
démontré par des études par PET utilisant un dérivé du glucose radioactif, le 18F-
fluorodéoxyglucose (129,277,278). Ainsi, plusieurs altération au niveau d cerveau orchestré par le
remodelage surviennent plusieurs années antérieurs au diagnostic.
Ces phénomènes représentent une forme de remodelage de la NVU commune à plusieurs
pathologies. Le remodelage de la NVU se traduit globalement par une réponse moléculaire et
cellulaire se produisant au sein de l’unité suite à un dommage (255). Toutefois, le rôle du
20
remodelage demeure controversé. Ce remodelage essaie de compenser pour l’événement stresseur
ce qui peut résulté en une amélioration ou aggravation la cascade pathogénique selon le contexte
(150). Dans la phase initiant la AD, l’hypoperfusion s’instaure et induit l’altération de la NVU qui
peut contribuer à l’initiation de la pathogenèse via un mécanisme peu connu (Section 1.3 –
Hypoperfusion) (93). Par la suite, un profil inflammatoire s’installe afin de promouvoir
l’élimination de l’Aβ et autres macromolécules toxiques. Toutefois, cette élimination n’est pas
efficace et, par conséquent, se prolonge ce qui contribue au dysfonctionnement neuronal. Des
investigations supplémentaires, s’intéressant au remodelage de la NVU et son effet sur les
pathologies, sont nécessaires et pourraient démystifier la mécanistique derrière la pathogenèse, en
plus d’offrir de nouvelles voies de traitement (53).
1.6. Hypothèses et objectifs
Dans le cadre de mon projet de recherche, nous nous sommes d’ailleurs intéressés à la relation entre
la maladie et le remodelage de la NVU, plus particulièrement au remodelage de la NVU à la suite
de l’hypoperfusion (Section 1.3 – Hypoperfusion) et de l’activité du t-PA (Section 1.4 – Système
de l’activateur du plasminogène). Puisque l’hypoperfusion et l’altération du t-PA sont des
comorbidités lié à l’âge et à la AD, nous supposons que ces deux conditions induisent de profonds
changements au sein de la NVU ce qui pourrait influencer la progression de la maladie.
Tel que décrit précédemment, une altération de l’autorégulation du CBF est associée à la AD
(118,123,124) indiquant une hypoperfusion cérébrale. Elle induit notamment une altération du
métabolisme du glucose (95,129,130) et contribue à la neurodégénération et au déclin cognitif
(93,95,130,134). Pour ces raisons, nous supposons que l’hypoperfusion constitue un mécanisme
précoce capital de la cascade pathologique de la AD tel que proposé par l’hypothèse vasculaire de
Zlokovic (42). La fonction des microglies étant hautement énergétique (279), nous présumons que
celle-ci deviendrait dysfonctionelle à la suite d’une hypoperfusion cérébrale modérée et ce, en
l’absence de mort cellulaire. Ainsi, nous estimons que l’hypoperfusion chronique, soit la réduction
permanente ou à long terme du CBF, induirait l’accumulation de l’amyloïde et d’autres débris
toxiques. Nous tenterons de confirmer cette hypothèse en nous concentrant sur la modulation des
cellules de la NVU, dont la microglie, lors d’une hypoperfusion chronique modérée. Pour ce faire,
nous avons développé un nouveau modèle jumelant une BCAO transitoire avec une 1VO
permanente.
Avec l’âge, l’expression du t-PA diminue perturbant ainsi l’homéostasie cérébrale. En effet, le
système du t-PA participe à plusieurs fonctions de maintenance du cerveau. Par exemple, le t-PA
21
contribue indirectement à la dégradation de l’amyloïde en convertissant le plasminogène en
plasmine (225,226) et en modulant, par son action de cytokine, l’activité des cellules
immunocompétentes (192–196). Le t-PA possède également un effet neuroprotecteur (185,197–
199,201) en plus d’un rôle anti-apoptotique chez les oligodendrocytes (175). De ce fait, nous
présumons qu’une compensation du t-PA retarderait la progression de la AD grâce à ces multiples
rôles (162,232). En effet, nous estimons que cette compensation favoriserait l’élimination de l’Aβ
ainsi que l’intégrité de la NVU. De plus, étant un ligand de LRP1 (222), nous estimons que le t-PA
peut moduler positivement l’ensemble des cellules de la NVU l’exprimant (cellules endothéliales,
microglies, péricytes, etc.). Ainsi, nous soupçonnons que l’injection d’une faible concentration du t-
PA en l’absence de bris de la BBB serait suffisante pour retarder la progression de la AD et ce, par
un mécanisme non enzymatique impliquant la voie LRP1. Nous tenterons alors de déterminer l’effet
de l’injection systémique faible et chronique (10 semaines) du t-PA sur la BBB, sur l’activité
microgliale, sur l’Aβ de même que sur les fonctions cognitives dans un contexte de la AD.
Nous cherchons, dans une première mesure, à déterminer comment l’altération de la NVU suite à
l’hypoperfusion peut influencer négativement la fonction cognitive et contribuer à la progression de
la AD. Par la suite, nous tentons de voir comment la modulation de la même unité par le système du
t-PA peut bénéficier et retarder la progression de la AD. Le présent projet décrit ainsi la
contribution du remodelage de la NVU dans la pathogenèse de la AD.
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Chapitre 2
2. Severe chronic cerebral hypoperfusion induces microglial dysfunction
leading to memory loss in APPswe/PS1 mice
Maude Bordeleau, BSc, Ayman ElAli, PhD, and Serge Rivest, PhD
Correspondence:
Dr. Serge Rivest
Neuroscience Laboratory
CHU de Québec Research Center (CHUL)
Department of Molecular Medicine, Faculty of Medicine, Laval University
2705 Laurier boulevard, Québec City
QC G1V 4G2, Canada.
Email: [email protected]
24
2.1. Résumé
La composante vasculaire de la maladie d’Alzheimer (AD) est un sujet de plus en plus étudié, ainsi
il a été proposé qu’une perturbation vasculaire initierait la neurodégénération dans la AD. En ce
sens, nous supposons qu’une hypoperfusion cérébrale chronique sévère (SCCH) perturbe l’activité
de l’unité neurovasculaire ce qui nuirait à l’élimination de l’amyloïde. Des souris APPswe/PS1 âgés
de 4 mois furent soumises à la SCCH. Après 15 semaines, les souris hypoperfusées démontraient
une altération de leur mémoire aux tests du labyrinthe en T et du 2-objets-nouvel-objet. Le déclin
cognitif corrélait avec une augmentation du nombre de plaques amyloïdes suggérant l’altération de
l’une des voies d’élimination de l’amyloïde. De ce fait, nous avons observé une perte de la fonction
des microglies. In vitro, nous avons démontré que ce phénomène était induit en condition faible en
glucose qui diminue l’activité globale, l’activation et la capacité phagocytique. Cette dysfonction
semble alors induire une diminution de l’efficacité d’élimination de l’amyloïde et des débris
neurotoxiques. De plus, la SCCH altère également la voie de ERK, soit la survie neuronale. En
contexte de la AD, la dysfonction de ERK et des microglies contribuent toutes les deux au déclin
cognitif. Ainsi, le développement de traitement ciblant l’hypométabolisme des microglies pourrait
rétablir la fonction microgliale et retarder la progression de la AD.
2.2. Abstract
The vascular components of Alzheimer's disease (AD) are the object of mounting interest in
the field. More precisely, disruption of vascular integrity has been hypothesized to be involved in
initiating the neurodegenerative cascade in AD. Vascular alterations associated to cerebral
hypoperfusion, impairs brain homeostasis, thus oxygen and glucose intake. Herein we suggest that
severe chronic cerebral hypoperfusion (SCCH) alters brain homeostasis, resulting in the alteration
of amyloid precursor protein processing. SCCH was induced in APPswe/PS1 mice for 15 weeks
later. SCCH worsened mice cognitive functions that were assessed by water T-maze and 2-object-
novel-object tests. Cognitive decline correlated with increased amyloid plaque abundance,
suggesting a dysfunction in amyloid-beta (Aβ) elimination. Indeed, SCCH impaired microglial cell
function, which are implicated in Aβ elimination. In addition, SCCH altered extracellular signal-
regulated kinases 1/2 (ERK1/2) pathway activity. Moreover, in vitro investigations showed that
microglia exposed to a low-glucose environment display a decreased global activity and phagocytic
capacity. Microglial cell dysfunction correlated with a lower efficiency in eliminating Aβ. Our
study unravels new insights into the implication of cerebral hypoperfusion in AD pathogenesis by
altering microglial cell activity, which should be considered while developing new therapies.
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2.3. Introduction
The most common form of dementia is currently Alzheimer’s disease (AD). According to current
estimates, over 90 million people will be affected in 2025 worldwide (2). AD is characterized by
two main hallmarks, i.e. senile plaques and neurofibrillary tangles (4) which are respectively caused
by deposition of amyloid-beta (Aβ) and tau hyperphosphorylation. In the last decade, a number of
studies have provided accumulating evidence of a close relationship between vascular risk factors
and AD (53,94,95,127,280). It was proposed that vascular dysfunction associated to cerebral blood
flow (CBF) alterations might trigger downstream events that are implicated in the
neurodegenerative cascade observed in AD (93,95). For example, low CBF has been associated
with mild cognitive impairment (MCI), a possible precursor in AD etiology (124,137,142,157).
Importantly, impaired CBF and glucose metabolism have been reported in AD patients (42), and in
mouse models of AD (140). More interestingly, deregulations in cerebrovascular perfusion have
been suggested to take place even before cognitive decline (144).
Recently, Zlokovic (2011) proposed the two-hit vascular hypothesis, which incorporated
cerebrovascular dysfunction as a key mechanism involved in AD pathogenesis (42). The hypothesis
speculates that several risk factors affecting vascular functions, such as hypertension, diabetes, heart
disease and cerebrovascular diseases, trigger blood-brain barrier (BBB) dysfunction initiating the
neurodegenerative cascade observed in AD (42). Accordingly, many studies have demonstrated
higher Aβ levels after cerebral hypoperfusion (140,144). Once Aβ starts to accumulate, soluble
oligomers may trigger neurotoxic and excitotoxic events (69). Aβ has also been shown to
profoundly affect the function of cells that form the neurovascular unit (NVU) (121). The NVU
comprises vascular cells (endothelial cells, pericytes), glia cells (astrocytes, microglia and
oligodendrocytes) and neurons (119,121). Importantly, the NVU plays a crucial role in maintaining
brain homeostasis and CBF (119,121,243,245,246). Recent investigations have shown that chronic
cerebral hypoperfusion (i.e. oligemia) impairs NVU function. Upon oligemia-induced injury, both
basal membrane thickening of endothelial cells and fibrous deposition occur (142). Hypoperfusion
is also well known to induce BBB dysfunction (55,121,149). Recent investigations have also
demonstrated altered astrocyte function upon hypoperfusion, leading to abnormal axon-glia
connections (141), characterized by the infiltration of astrocyte branches in neuronal cell bodies
(156). Importantly, the latter phenomenon is known to promote neuronal apoptosis (156).
Furthermore, a few studies have observed altered numbers of astrocytes (157,281), neurons
(149,151,154–156), microglia (151,156,281) and peripheral monocytes (126) following either
chronic or acute hypoperfusion. Importantly, the brain possesses several sophisticated mechanisms
26
involved in Aβ clearance, namely degradation by microglia (282,283). However, Aβ clearance by
microglia is impaired in the advanced stages of AD due to the formation of a stressful
microenvironment (284). Importantly, how chronic cerebral hypoperfusion may affect the function
of microglia remains totally unknown (53).
In the light of previous studies, we hypothesize that severe chronic cerebral hypoperfusion induces
NVU dysfunction, thereby impairing brain homeostasis, thus altering Aβ clearance and
exacerbating neurodegeneration. We have developed a new model of severe chronic cerebral
hypoperfusion (SCCH) (Fig. 2.1) to study the changes occurring within the NVU and the
mechanisms that contribute to the progression of AD pathogenesis. Interestingly, SCCH worsened
mouse spatial and non-spatial memory loss. In addition, we found that SCCH likely triggers the
formation of a low glucose environment, thus reducing microglial cell efficiency of in eliminating
Aβ. In addition, we also report that SCCH deactivates extracellular signal-regulated kinase (ERK)
pathway, which have been shown to play an important role in neuronal survival. Overall, SCCH
exacerbated AD-like pathology in APPswe/PS1 mice, thus confirming oligemia as an event that can
significantly contribute to neurodegeneration.
2.4. Material and methods
2.4.1. Animals with severe chronic cerebral hypoperfusion
All protocols were performed according to the Canadian Council on Animal Care guidelines, under
supervision by the Laval University Animal Welfare Committee. Mice were acclimatized to
standard laboratory cycles of 12 h light/dark (on at 7:00 h and off at 21:00 h). Adult male
APPswe/PS1 transgenic mice expressing a doubly mutated version of human amyloid precursor
protein (APPswe) as well as human presenilin 1 (A246E variant) were acquired from the Jackson
Laboratory (Bar Harbor, ME). All mice had a C57BL/6J background.
Four months-old APPswe/PS1 mice were subjected to either sham or SCCH surgery. Mice were
anesthetized for 5 min with 3.0% isoflurane (Abbvie, Chicago, IL), and anesthesia was further
maintained using 2.5% isoflurane for ~15 min. Lidocaine was injected locally before performing
incisions. Midline cervical incision was performed and both common carotid arteries were then
exposed. The right common carotid artery was permanently ligated with 6.0-mm non-absorbable
silk thread as described in Pimentel-Coelho et al. (2013) (138). After 2 min, a pressure of 15 g/mm2
was applied on the left common carotid artery with a vessel clamp (MORIA Inc, Doylestown, PA)
during 15 sec (Fig. 2.1). Midline incision was closed using 6.0-mm silk suture thread, and mice
were awakened using a heating pad. Buprenorphine was administered 12 h post operation. Body
27
mass time course after surgery was monitored for eight weeks. Mice were kept for 17 weeks after
surgery.
At 7.5 months, mice were subjected to behavioral analysis. Three weeks later, they were
anesthetized with ketamine/xylazine, and total blood was collected followed by flush perfusion with
saline (0.9% (w/v) NaCl). Brain was split in two halves at the origin of the hippocampus ( -1.0 mm
from the bregma). The anterior part of the brain, along with the midbrain, was post-fixed with 4%
(w/v) paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA) in 0.1 M phosphate
buffer (pH = 7.6) for 48 h and immersed in 20% (w/v) sucrose/4% (w/v) PFA overnight. The
posterior part of the brain was frozen in dry ice and cut into 25-µm coronal sections with a
microtome (Leica SM 2000R, Leica Microsystems Inc., Concord, ON, Canada). Tissues were
stored at -20 °C in a cryoprotectant solution (0.052 M sodium phosphate buffer (pH = 7.3)
containing 5.37 M ethylene glycol and 2.71 M glycerol), which was used for immunofluorescence,
Fluoro Jade-B (FJB) staining, Nissl body staining and immunohistochemistry.
Immunohistochemistry and FJB staining were carried out in parallel in stroked animals as positive
controls for loss of BBB integrity and monocyte infiltration. The anterior part of the brain was used
for the molecular analyses (vide infra).
Figure 2.1. Schema illustrating the SCCH surgery. Permanent occlusion of the right common carotid
artery is performed on anesthised mice. After 2 minutes, a 15G pressure is exerted with a vessel clip on
the left common carotid artery during 15 seconds.
28
2.4.2. Behavior analysis
Behavior analysis was carried out 14 weeks post surgery. All behavior tests were performed during
the lights-off phase of the day. The behavioral experimenter was blinded to the surgery status of the
animals.
2.4.2.1. Water T-maze
Hippocampal-dependent spatial learning and memory were determined via the water T-maze test as
previously described (285). The mouse’s ability to find a submerged platform in a water-filled
fiberglass pool (stem length, 64 cm; arm length, 30 cm; width, 12 cm; wall height, 16 cm) was
evaluated. Before the test, the escape platform (11 cm x 11 cm) was placed randomly at the end of
one of the arms and submerged to a 1-cm depth. Mice were put in the stem's enclosure and let to
explore the maze until the animal escaped to the platform. If the mouse did not find the submerged
platform after 60 sec, it was gently maneuvered towards it. The animals stayed on the platform for
20 sec. During the learning and reversal phase, the time and number of trials required to reach the
platform were noted. The criterion of positive learning was determined as successfully reaching the
platform upon 5 consecutive trials; otherwise, mice had 48 trials at their disposal to achieve
learning. Fourty-eight hours later, the reversal-learning phase was assessed by placing the
submerged platform at the opposite end of the maze
2.4.2.2. Two-object novel object recognition
The object recognition test consisted of training and testing phases in a rectangular open field of the
same size as their home cage (width, 18 cm; length, 28 cm; height, 12 cm) in clear plexiglas. The
experimenter could not be seen by animals while the test took place. During the training phase,
mice were presented with two identical objects and allowed to inspect both of them for 5 min.
Animals whose exploration period lasted < 10 sec per item were considered unsuitable and were not
used for further analysis. Between each trial, objects were thoroughly cleaned to minimize olfactory
cues. After 3 h, the retention test was performed. Mice were left in the experimental arena for 5 min
in the presence of a familiar object along with a new one of comparable size, texture and shape. For
half of the animals, the new object was presented on the right-hand side and for the remaining half,
on the left-hand side. A digital camera was mounted on the ceiling above the arena and connected to
a computer equipped with a video tracking system (ANY-maze, Stoelting Co., Wood Dale, IL),
which objectively monitored and quantified movements. Object exploration was defined as
touching the object or pointing the snout towards it at a distance < 2 cm. The recognition value
represents ratio of the time spent exploring the object on the total time allowed.
29
2.4.2.3. Asymmetry cylinder test
Mice were subjected to a single 5-min session within a glass cylinder (diameter, 20 cm; height, 30
cm). An angled mirror was placed behind the cylinder in order to visualize limb use and movements
from all angles. During the session, forelimb asymmetry was assessed by scoring independent
weight-bearing contacts of the right or left paw on the cylinder wall. The percentage of right and
left touches was calculated relative to the total number of contacts.
2.4.2.4. Open field
Mice were placed in a rectangular open-field arena for 5 min as previously described (286) and
equipped with a video tracking system (side view). The distance traveled, the frequency and time of
the motionlessness episode, the frequency and direction of rotation (clockwise or counterclockwise)
as well as maximum speed reached were recorded. The time spent and entries in the different parts
of the arena (center, interspace and periphery) were also recorded. After each trial, fecal waste was
removed, and the floor of the arena was cleaned with a damp cloth and then dried.
2.4.3. Soluble Aβ1-40 and soluble Aβ1-42 ELISA
The soluble Aβ1-40 and soluble Aβ1-42 ELISAs were performed according to the manufacturer’s
protocol (EMD Millipore, Billerica, MA). Contralateral and ipsilateral hemispheres were split and
homogenized in 750 µL of lysis buffer containing a protease inhibitor cocktail (EMD Millipore)
and 1% (v/v) phosphatase inhibitor cocktail 3 (Sigma-Aldrich, St. Louis, MO) with a hand-held
homogenizer (Bio-Gep Series Pro200, Pro Scientific, Oxford, CT). A sample of the homogenized
brain solution was collected for western blot analysis. The remaining solution was mixed at 4 °C for
2 h. Brain homogenates were spun for 10 min x 1300 rpm at 4 °C and the supernatant was collected.
Fifty µL of the diluted samples (1:10) and standards (16-500 pg/mL) were loaded along with 50 µL
of antibody conjugate per well into ELISA plates. Samples were mixed for 5 min at 4 °C and then
stored overnight at 4 °C without agitation. The wells were rinsed five times with washing buffer
prior to incubation with conjugate-enzyme for 30 min at room temperature (RT). The wells were
rinsed again before substrate addition. ELISA plates were incubated for 30-35 min at RT and the
binding reaction was ended with stop solution containing 0.3 M HCl. Soluble Aβ1-40 and soluble
Aβ1-42 levels were measured by the A495 and A590 values using a microtiter plate reader (SpectraMax
340PC, Molecular Devices, Sunnyvale, CA) and analyzed using the SOFTmax Pro3.1.1 software
(Molecular Devices).
30
2.4.4. Immunofluorescence staining
Brain sections were stained using a free-floating technique. Tissues were rinsed 3 times for 15 min
in potassium phosphate buffered saline (KPBS) containing 22 mM K2HPO4, 3.3 mM KH2PO4 and
140 mM NaCl. Sections were then permeabilized and blocked in 6.78 mM Triton X-100, 1% (w/v)
BSA (Sigma-Aldrich) and 4% (v/v) goat serum (Cedarlane, Burlington, ON, Canada) for 20-30
min. Tissues were incubated overnight at 4 °C with the primary antibody in 0.5X blocking solution.
We used mouse anti-Aβ monoclonal antibody (6E10) (Wako Chemicals, Richmond, VA), rabbit
anti-ionized calcium binding adaptor molecule 1 (Iba1) antibody (Wako Chemicals) and rat anti-
CD68 (AbD Serotec, Kidlington, UK) as primary antibodies. Free-floating slices were next rinsed
in KPBS and incubated with the secondary antibody for 2 h at RT. Tissues were incubated with
Cy3-conjugated goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA), Alexa
488-conjugated goat anti-rabbit antibody (Life Technologies, Burlington, ON, Canada) or Cy3-
conjugated goat anti-rat antibody (Jackson ImmunoResearch). Brain sections were washed and
counterstained with 2 µg/mL DAPI (Life Technologies) for 20 min at RT. Sections were rinsed,
mounted onto SuperFrost slides and dried under vacuum for at least 3 h. Brain mounted slides were
hydrated and overlaid with coverslips using iFluoromount-G anti-fading medium (Electron
Microscopy Sciences).
For cellular stereological analysis, Iba1 was co-stained with either 6E10 or CD68, using the Iba1
antibody as the initial marker in the staining sequence, followed by the 6E10 or CD68 antibody.
Next, three sections for each Iba1/6E10- (-1.46 mm, -2.18 mm and -3.28 mm from the bregma) and
Iba1/CD68-stained tissue (-1.46 mm, -2.06 mm and -2.46 mm from the bregma) were analysed
using Stereo Investigator v. 9.10.6 (MBF Bioscience, MicroBrightField Inc., Williston, VT). For
each section, amyloid load (number and area), microglia number and CD68 marking (number and
area) were determined per hemisphere and expressed as ratios of the hippocampus area. Pictures
were taken with a Nikon C80i microscope (Nikon Instruments, Williston, VT) equipped with a
QImaging® color camera (MBF 2000 R, Quantitative Imaging, Surrey, BC, Canada) and QCapture
Version 2.98.2 software (Quantitative Imaging).
2.4.5. Western blot analysis
Whole brain homogenates were diluted 1:10 in NET lysis buffer (0.2 M NaCl, 0.1 M Tris, 5 mM
EDTA, 11.6 mg/L Tergitol® solution (232mg/L) type NP-40, pH = 7.5) containing 1% (v/v) of
phosphatase inhibitor mixture and 1% (v/v) of protease inhibitor cocktail (Sigma-Aldrich) and
sonicated for 40 sec on ice (Sonic Dismembrator, model 100, Fisher Scientific Ca., Ottawa, ON,
Canada). Total protein content for each sample was determined using the bicinchoninic acid method
31
(QuantiPro assay kit, Sigma-Aldrich) (287). Protein samples (15 µg) were mixed with 2X SDS
loading buffer and heated for 5 min at 100°C. Samples were loaded on a precast 4-20%
polyacrylamide gradient gel (Bio-Rad, Hercules, CA) and subjected to electrophoresis for 10 min at
110 V followed by 90 min at 90 V. After migration, resolved protein bands were transferred onto a
0.45-mm polyvinylidene fluoride (PVDF) membrane (EMD Millipore) for one hour on ice under a
80 V potential. The PVDF membrane was rinsed three times with a 0.1 M Tris-buffered saline
containing 670 M Tween-20 (TBS-Tween; Sigma-Aldrich) and blocked in TBS-Tween with 5%
(w/v) skim milk for 30 min at RT. The PVDF membrane was then incubated overnight at 4 °C one
of the following primary antibodies: anti-phosphoERK1/2 (T202/Y204 for ERK1, T185/Y187 for
ERK2; Cell Signaling Technology, Danvers, MA), anti-total ERK1/2 (Cell Signaling Technology),
anti-phospho-p38 (New England Biolabs, Whitby, ON, Canada), anti-total p38 (New England
Biolabs), anti-phospho-stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK; New
England Biolabs), anti-total SAPK/JNK (New England Biolabs), anti-claudin-5 (Santa Cruz
Biotechnology, Dallas, TX) and anti-β-actin (EMD Millipore), the latter being used as an internal
standard for sample processing errors. The membrane was washed three times with TBS-tween,
incubated for two hours at 4 °C with the horseradish peroxidase-conjugated secondary antibody in
blocking solution, and re-washed three times with TBS-tween. Proteins were detected by enhanced
chemiluminescence (ECL; GE Healthcare Life Sciences, Mississauga, ON, Canada) (288) and
exposed on BioMax® MR film (Carestream, Rochester, NY). Blots were digitized and analyzed by
densitometry with ImageJ software (National Institutes of Health, Bethesda, MD). Specific protein
levels were expressed relative to the loading control (β-actin internal standard).
2.4.6. Flow Cytometry
During perfusion, cardiac puncture was performed to quantify monocytes in peripheral blood using
flow cytometry analysis. Blood was stored in EDTA-coated vials (Sarstedt, Newton, NC). Cells
were washed once with Ca2+/Mg2+-free Dulbecco’s PBS (DPBS; Sigma-Aldrich), and then
resuspended and incubated in DPBS supplemented with purified rat anti-mouse CD16/CD32
antibody (Mouse BD Fc Block; BD BioSciences) for 15 min at RT. Cells were then washed with
DPBS, resuspended in a mixture of V500-conjugated anti-CD45 antibody (BD BioSciences),
Alexa700-conjugated anti-CD11b antibody (eBioScience, San Diego, CA), allophycocyanin-
conjugated anti-CD115 antibody (eBioScience), phycoerythrin-conjugated anti-Ly6-G antibody
(BD Biosciences) and V450-conjugated anti-Ly6-C antibody (BD BioSciences). After a 40-min
incubation on ice, cells were rinsed with DPBS. Red blood cells were lysed with BD Pharm LyseTM
(BD Biosciences) for 30 min at RT. Labeled cells were washed, resuspended in DPBS, and then
32
injected in a flow cytometer (BDTM LSR II, BD Biosciences). A minimum of 67,000 singlet events
were acquired and analyzed with BD FACSDivaTM software v.6.1.2 (BD Biosciences)
2.4.7. Immunohistochemistry
Free-floating brain sections were washed with KPBS three 10-min cycles and incubated for 2 h at
RT with the appropriate secondary antibody, i.e. either biotinylated anti-goat antibody (Vector
Laboratories, Burlingame, CA) or biotinylated anti-rat antibody (Vector Laboratories). Brain slices
were washed, treated with avidin/biotin complex reagent (Vector Laboratories) for one hour at RT
and washed again. Albumin and CD45 were stained in 0.5 g/L diaminobenzidine (DAB; Sigma-
Aldrich) solution supplemented with 1.11 mM H2O2 for 8 min and 15 min, respectively. Sections
were immediately washed, mounted onto SuperFrost slides, and dried overnight. Slides were
dehydrated using a sequential treatment with 10 dips in H2O, 50% (v/v) EtOH, 70% (v/v) EtOH,
twice in 95% (v/v) EtOH, thrice in 100% (v/v) EtOH, and finally twice in xylene. The dehydrated
sections on slides were overlaid with coverslips using distyrene plasticizer xylene (DPX) mounting
medium
IgG extravasation staining was performed as described above when used as a secondary antibody.
Free-floating brain slices were washed and treated with a blocking solution (6.78 mM Triton X-100,
1% (w/v) BSA and 4% (v/v) horse serum). Biotinylated anti-mouse IgG antibody was incubated
overnight at 4°C. Slices were rinsed with KPBS and immersed for 1 h with ABC mixture. Brain
sections were washed, stained in DAB solution for 10 min, and re-washed. Slices were mounted
onto slides, dried, dehydrated and overlaid with coverslips in DPX. Pictures were taken using a
Nikon C80i microscope equipped with a QImaging® color camera.
2.4.8. Nissl body staining
Brain sections were mounted on slides and dried overnight under vacuum. Slides were washed
(2x10 min) in KPBS, and then fixed for 20 min in 4% (w/v) PFA. They were then dehydrated
according to the following sequence: H2O, 50% (v/v) EtOH, 70% (v/v) EtOH, twice in 95% (v/v)
EtOH, and thrice in 100% (v/v) EtOH; each dip carried out for 3 min), transferred next into xylene
for 5, 30 and 2 min, and finally rehydrated using the reverse-order sequence (thrice in 100% (v/v)
EtOH, twice in 95% (v/v) EtOH, and then 70% (v/v) EtOH, 50% (v/v) EtOH and H2O; 2 min for
each dip). Slides were next dipped 20 times in 0.25% thionin (Sigma-Aldrich). Mounted brain
slides were dehydrated again (20 dips in each of H2O, 50% (v/v) EtOH, and 70% (v/v) EtOH, and
then 2 and 3 cycles of 3-min dips in 95% (v/v) and 100% (v/v) EtOH, respectively, and finally, 2
33
cycles of 3-min dips in xylene) before overlaying with coverslips in DPX mounting medium
(Electron Microscopy Sciences).
For stereological analysis, 3 sections (-1.46, -2.06, and -2.46 mm from the bregma) were analyzed
using the Stereo Investigator software. For each section, the cornu ammonis 1 through 3 (CA1/CA2,
CA3) areas and the dentate gyrus area were defined and expressed as ratio of total hippocampus
area for both hemispheres. Photographs were obtained with a Nikon C80i microscope equipped
with a QImaging® color camera.
2.4.9. Fluoro-Jade B staining
FJB staining was used as an indicator of neuronal death as described in Turrin and Rivest (2006)
(289). After mounting brain sections onto slides and thorough overnight drying under vacuum,
slices were fixed with 4% PFA for 20 min. Fixed slides were then rinsed twice with KPBS for 5
min, before a cycle of dehydration/rehydration according to this sequence: 3 min in 50% (v/v)
EtOH, 1 min in 70% (v/v) EtOH, 3 min in 100% (v/v) EtOH, 1 min in 70% (v/v) EtOH, 1 min in
50% (v/v) EtOH and 1 min in H2O). Mounted slides were next treated for 10 min with 3.8 mM
potassium permanganate (MP Biomedicals, Santa Ana, CA), rinsed for 1 min with H2O and then
incubated in 6.05 µM FJB solution (EMD Millipore) containing 17.5 mM acetic acid and 2 µg/mL
DAPI. Then, slides were washed (3 x 1-min rinses in H2O) and dried overnight. Slides were
immersed in xylene (3 x 2-min dips) and overlaid with coverslips in Fluoromount-G anti-fading
mounting medium. Images were obtained with a Nikon C80i microscope equipped with a
QImaging® color camera.
2.4.10. In vitro experiments
2.4.10.1. Cell culture
Immortalized murine microglial cells (BV2) were cultured at 37 °C in a 5% CO2/95% air
atmosphere in DMEM containing 10% (v/v) FBS, 4.5 g/L glucose, 6 mM L-glutamine, 100 U/mL
penicillin and 100 µg/mL streptomycin. Cells were grown to 70-90% confluence and subjected to a
maximum of 20 passages. All cell culture reagents were from Sigma-Aldrich.
2.4.10.2. XTT assay
Cell viability was measured with the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-
carboxanilide (XTT) assay according to the manufacturer’s procedure (Cell Signaling Technology).
BV2 cells were plated at 1x104 cells/well in 96-well microplates in DMEM supplemented with 10%
(v/v) FBS and 4.5 g/L D-glucose. Cells were incubated overnight at 37°C before replacing medium
with DMEM containing 1% (v/v) FBS and either 4.5 g/L D-glucose (DMEM-High) or 1.0 g/L D-
34
glucose (DMEM-Low; n=5 experiments, 4 replicates/treatment). For cell death controls, cells were
exposed for 24 h to DMEM-High containing 0.75 mM H2O2). Fifty µL of XTT detection solution
were added to each well and incubated for 3 h at 37 °C. Cell viability was determined from A450
using a microtiter plate reader and analyzed with the SOFTmax Pro3.1.1 software.
2.4.10.3. Griess assay
Global cell activity was evaluated by determining nitrite release into the cell culture medium using
the Griess assay according to the manufacturer’s protocol (Life Technologies). Cells (1.8 x
104/well) were plated in 24-well culture plates and incubated overnight at 37°C. BV2 cells were
incubated for 24 h in both DMEM-High or -Low medium plus or minus lipopolysaccharide (LPS)
(Sigma-Aldrich) (at either 0.1, 0.5 or 1 µg/mL; n=6 experiments, treatments in triplicate). Culture
medium was then collected, and 150 µL were transferred to 96-well microplates. Nitrite-containing
medium was mixed with 20 µL of Griess reagent and 130 µL of deionized water. Standards (1-100
µM) were added to the microplate, which was next incubated for 30 min at RT. Nitrite
concentration was determined by measuring A508 with a microtiter plate reader and analyzed with
the SOFTmax Pro3.1.1 software.
2.4.10.4. Flow cytometry analysis of CD68 expression
BV2 cells were plated (3x105 cells/well) in 24-well culture plates in DMEM containing 10% (v/v)
FBS and 4.5 g/L D-glucose. Cells were allowed to grow overnight at 37°C with 5% CO2 before
replacing medium with DMEM-High or -Low (n=5 for each group). After a 24-h incubation, cells
were harvested upon treatment with cell dissociation buffer (Sigma-Aldrich) and transferred to 5-
mL FACS tubes (BD Biosciences). Cells were spun down for 10 min at 300 x g and resuspended in
DPBS containing 4% rat serum (Jackson ImmunoResearch). Cell metabolism was stopped by
storage for 20 min on ice, and cells were then collected by centrifugation, washed and further
incubated in 0.5 g/L Fc Block CD16/CD32 on ice for 20 min. Cells were next washed once with
DPBS and suspended in 100 µL of DPBS containing 0.5 g/L Alexa 647-conjugated anti-CD68
antibody (AbD Serotec), and incubated on ice for 30 min. After one rinsing with DPBS, cells were
resuspended in DPBS. Data were acquired using a flow cytometer (at least 10,000 singlet events)
and analyzed with BD FACS Diva software.
2.4.10.5. Flow cytometric analysis of phagocytosis
BV2 cells were plated at 3x105 cells per well in DMEM containing 4.5 g/L D-glucose and 10%
(v/v) FBS, and incubated at 37°C overnight. DMEM was next replaced with DMEM-High or -Low
(n=6 for each group). Cells were acclimatized in the latter medium for 24 h before adding 5 mg/L of
35
Vybrant E. coli beads (Life Technologies) in 1X Hank's balanced salts solution. Phagocytosis was
allowed to proceed for 2 h at 37 °C. As a control, phagocytosis was assessed on ice with viable
cells. Cells were then washed twice with ice-cold DPBS and harvested with non-enzymatic cell
dissociation buffer (Sigma-Aldrich). Non-internalized beads were quenched with 0.2% trypan blue
solution (Sigma-Aldrich). BV2 cells were washed and resuspended in DPBS, and then injected into
the flow cytometer. Data were acquired (at least 500 singlet events) and analyzed with the BD
FACS Diva software.
2.4.11. Statistical analysis
In vivo and in vitro data are expressed as the mean ± SEM and mean ± SD, respectively. For cellular
and molecular analyses, comparisons between groups were analyzed with standard two-tailed
unpaired t-tests. For weight variation through time, the intergroup differences were calculated by
two-way ANOVA. Differences were considered as significant with a P value < 0.05. All statistical
analyses were performed using GraphPad Prism v.6.0 for Windows (GraphPad Software Inc., La
Jolla, CA).
2.5. Results
2.5.1. SCCH worsen memory impairment in APPswe/PS1 mice without affecting motor
capacity
Weight variation in both groups was monitored for up to 7 weeks after surgery, which had no effect
per se on weight gain (Fig. 2.2A). Water T-maze and two-object novel object recognition tests were
performed 14 weeks after surgery to distinguish changes in hippocampal-dependent spatial memory
and learning (285) from alterations in non-spatial memory (290), respectively. The non-spatial
memory or recognition memory is based on the novelty paradigm which postulates a subject's
preference to explore a new object rather than a familiar one (290). For the water T-maze test,
SCCH-treated compared to sham-operated APPswe/PS1 performed likewise during the acquisition
phase (data not shown). Both groups displayed a latency of ~11 sec and tried approximately 7
times. However, 48 hours later, during the reversal phase, SCCH mice had significantly (P=0.0350)
higher latency (Fig. 2.2C) and needed more trials to complete the test (P=0.0681) (Fig. 2.2B). Both
groups performed likewise in the learning phase of the two-object novel object recognition test
(data not shown), while SCCH mice showed a significant loss of the novelty paradigm in the
retention phase compared with sham (P=0.0243) (Fig. 2.2D). Sham and SCCH mice explored the
objects 48.62-51.58% and 48.17-51.83% of the time, respectively.
36
Motor performance was assessed with open-field and asymmetry tests, which respectively define
motor capacity and laterality tendency. Sham and SCCH mice behaved likewise for both tests
(Supplementary Fig. 2.1). During the open-field test, both groups showed equivalent distance
traveled (sham: 33.3 ± 1.3 cm, SCCH: 27.4 ± 2.4 cm), immobile time (sham: 27.1 ± 4.2 sec, SCCH:
49 ± 14 sec), immobile episodes (sham: 16.7 ± 3.1, SCCH: 23.9 ± 4.2), maximum speed (sham:
0.320 ± 0.015 cm/s; SCCH: 0.327 ± 0.021 cm/s), as well as the number of total rotations (sham:
19.5 ± 0.3; SCCH: 16.5 ± 2.5), clockwise rotations (sham: 9.3 ± 1.2, SCCH: 8.5 ± 1.7) and
anticlockwise rotations (sham: 10.2 ± 1.1, SCCH: 8.0 ± 1.5). (Supplementary Fig. 2.1A). In the
asymmetry cylinder test, sham and SCCH mice used their right forepaw 50.8 ± 2.1% and 45.5 ±
2.7% of the time, respectively (Supplementary Fig. 2.1B). Hence, chronic hypoperfusion did not
Figure 2.2. SCCH aggravates APPswe/PS1 memory loss. Water t-maze test (B,C) and 2-objects novel
object recognition task (D) respectively use to describe spatial memory and cognition. The surgery had no
significant effect on weight variation (A) between sham and SCCH group. SCCH mice present a tendency
(P = 0.0681) to increase trials number (B) and significant increase of latency (C) during water t-maze test.
SCCH group also exhibits significant loss of novel object recognition. Data are means ± SEM (n=6-8
animals per group) * P < 0.05 compared to sham group; #P < 0.05 compared with sham object 1. Pre-op:
Pre-operatory.
37
elicit any significant asymmetry pattern. Taken together, these results show that SCCH specifically
affects spatial and non-spatial memory without altering motor abilities in APPswe/PS1.
2.5.2. Memory loss in SCCH mice is associated with an increased number of parenchymal
amyloid plaques
Aβ deposition in brain parenchyma and vasculature constitutes one of the main hallmarks of AD
(4). Soluble Aβ oligomers have been described as neurotoxic species whereas the role of senile
plaques in the progression of the disease is still debated (69,80). Therefore, we evaluated Aβ load in
the hippocampus by using 6E10 immunostaining (Fig. 2.3A-C) and measured soluble Aβ1-40 and
Aβ1-42 levels by ELISA (Fig. 2.3D-E) . Our main interest resided in the hippocampus since the latter
has been suggested to be especially vulnerable to chronic cerebral hypoperfusion (126).
Stereological analysis of 6E10 staining revealed a significantly increased number of Aβ plaques in
SCCH mice compared to sham mice (P= 0.0008) (Fig. 2.3A, B) without a significant change of
plaques load (Fig. 2.3A, C). There was no difference in soluble Aβ1-40 (Fig. 2.3D) and Aβ1-42 (Fig.
2.3E) levels between sham and SCCH group. Interestingly, these results suggest that SCCH
APPswe/PS1 mice develop more amyloid plaques in the parenchyma without altering levels of
soluble oligomers.
2.5.3. SCCH-linked trend towards an increased in the patrolling monocyte population
Monocytes are known to be recruited to vasculature following injury (282,283) and to participate in
Aβ elimination (284,285,290). Thus, we investigated the effect of SCCH on peripheral blood
monocytes by using flow cytometry analysis. Leukocyte populations were gated with CD45+ in
which CD11b+/CD115+ represented monocyte populations (Fig. 2.4A). Via a gating strategy that
uses Ly6C as a cell marker (Fig. 2.4B), monocyte subsets were sorted into Ly6CHigh (Fig. 2.4C) and
Ly6CLow (Fig. 2.4D), corresponding to inflammatory and patrolling monocytes, respectively. Both
sham and SCCH mice exhibited similar monocyte and Ly6CHigh population frequencies. However,
the frequency of Ly6CLow showed a slight increase upon SCCH (P < 0.0779). The latter trend
towards a small shift in the Ly6CLow monocyte population may be caused by the vascular
dysfunction associated to SCCH, as these cells have been reported to actively contribute to vascular
remodeling (291–294).
Next, we examined the effects of SCCH on BBB integrity.. The integrity of the BBB was evaluated
by the extravasation of IgG and albumin, as well as by tight junction protein expression, using
immunohistochemistry (Supplementary Fig. 2.2A, B) and Western blot analysis (Supplementary
Fig. 2.2C), respectively. We chose claudin-5 as it is a major tight junction protein at the BBB,
38
which has been previously reported to decrease after bilateral carotid artery stenosis (BCAS)
(281,295). Unlike stroke brains, the brains of sham and SCCH mice showed no extravasation of
either IgG (Supplementary Fig. 2.2A) or albumin (Supplementary Fig. 2.2B) Likewise, no
significant change in claudin-5 protein levels was observed (Supplementary Fig. 2.2C). We next
examined whether SCCH might induce an infiltration of leukocytes into the brain. Neither sham nor
SCCH mice showed any evidence of leukocyte infiltration, as shown by CD45
Figure 2.3. Number of amyloid plaques increase following SCCH without any change in amyloid
burden. Amyloid burden in APPswe/PS1 is characterized by immunofluorescence staining (A-C) and
ELISA (D,E). SCCH group has more plaques than sham group (A,B) without significant changes in the
plaque load (C). Same levels of soluble Aβ1-40 (D) and soluble Aβ1-42 (E) are observed in both groups. Data
are means ± SEM (n=6-8 animals per group, 3 sections per animal’s brain for immunofluorescence staining)
*** P < 0.005 compared with sham. Images were acquired with 4X objective. Scale bar = 500µm.
39
immunocytochemistry (Supplementary Fig. 2.3). In contrast, stroke injury showed monocyte
recruitment at the lesion site, as expected (Supplementary Fig. 2.3) . Thus, SCCH does not disrupt
BBB nor induce detectable immune cell infiltration, indicating that SCCH did not affect the
physical properties of the BBB.
2.5.4. SCCH disrupts plaque coverage by microglia and alters microglial activation
Being part of the NVU, we next assessed the implication of NVU dysfunction associated to SCCH
in affecting the function of microglia, which have been shown to be implicated in Aβ clearance
(284). Microglial coverage, immunoreactivity as well as CD68 expression that reflects microglial
cell activity state, were evaluated by stereological analysis of immunofluorescence staining (CD68,
Iba1 and 6E10; Fig. 2.5). Interestingly, SCCH mice exhibited significantly lower microglial Aβ
plaque coverage compared to sham mice (P=0.0108) (Fig. 2.5A). Following SCCH, microglia
throughout the brain also appeared to be less CD-68-immunoreactive, albeit at a weaker
significance threshold (P=0.1031) (Fig. 2.5B). However, in the vicinity of Aβ plaques, the number
of CD-68-immunoreactive microglia significantly decreased (P=0.0075) (Fig. 2.5C) and the area
Figure 2.4. Tendency of increased patrolling monocytes is observed following SCCH. Frequency of
total (A), inflammatory (C) and patrolling monocytes (D) were analysed by FACS on blood. Using a gating
strategy (B), inflammatory and patrolling were discriminated. SCCH does not influence monocyte frequency
in leukocytes (CD45+) (A), neither inflammatory monocyte (Ly6CHigh) frequency in total monocytes (C).
Patrolling monocyte (Ly6CLow) frequency seems (P = 0.0779) to increase (D). Data are means ± SEM (n=6-
8 animals per group). Data were analysed with standard two-tailed unpaired t- test’s.
40
covered by CD68-immunoreative microglia slightly decreased in SCCH mice (P=0.0559) (Fig.
2.5D) . The latter data indicate that alteration in microglial coverage and activation pattern promotes
the accumulation of amyloid deposition.
2.5.5. Alteration of microglial function is caused by an impaired glucose metabolism
To understand the mechanism underlying the alterations observed in microglial function, we used
an in vitro approach by maintaining immortalized microglia cell line BV2 in a low-glucose
environment that reflects, to some level, glucose metabolism impairments in vivo. Using an XTT
assay, we demonstrated that the low-glucose medium (DMEM-Low) had no effect on cell viability
(Fig. 2.6A) or cell proliferation (DMEM-High, 3 h: 1.57 ± 0.05, DMEM-Low, 3 h: 1.59 ± 0.10,
DMEM-High, 24 h: 2.18 ± 0.15, DMEM-Low, 24, h: 2.28 ± 0.15; data not shown) compared to
normal medium (DMEM-High). Between 3 and 24 h, BV2 growth significantly increased in
DMEM-High (P=0.0046) and DMEM-Low (P=0.0057). Moreover, microglial proliferation was
similar in both media. Thus, a low-glucose environment has no effect on microglial viability and
proliferation.
Next, global activity, activation and phagocytic capacity of microglia were assessed. Global activity
was determined by nitrite production when BV2 were exposed to increasing concentrations of
lipopolysaccharide (LPS). No change in nitrite production was detected in the absence of LPS (Fig.
2.6B) . BV2 cells in DMEM-Low produced significantly less nitrites than in normal condition in the
presence of 0.1 µg/mL LPS (P = 0.0022; Fig. 2.6B), 0.5 µg/mL (P = 0.0112; Fig. 2.6B) and 1
µg/mL (P = 0.0321; Fig. 2.6B) . Activated microglia (CD68+) were evaluated by flow cytometry
(Fig. 2.6C). When compared to microglia incubated in DMEM-High, suboptimal glucose
concentrations significant decreased the activation of microglia (P=0.0003). Using fluorescent
beads, a flow cytometry-based phagocytosis assay was performed with BV2 cells previously
exposed to DMEM-High or -Low for 24 h. There was significant fluorescent bead intake under both
conditions compared to control (P< 0.0001). Bead phagocytosis by BV2 cells significantly
decreased when cells were incubated with low-glucose medium (P < 0.0001) (27.05 ± 1.511%; Fig.
2.6D) compared to incubation with a normal glucose concentration (53.73 ± 1.353%; Fig. 2.6D) . In
view of the altered microglial activation observed in vivo upon hypoperfusion, the latter results
suggest that a low-glucose environment may disrupt microglial functions such as activation and
phagocytic capacity.
41
Figure 2.5. SCCH alters microglial function in APPswe/PS1. Microglial function is characterized by
immunofluorescence staining (A-E). SCCH significantly decreases microglial coverage (Iba+) (B) and
number of activated microglia (CD68+) in the vicinity of amyloid plaques (D). SCCH has a tendency (P =
0.0559) to decrease area of activated microglia locally to amyloid plaques (E). Microglial
immunoreactivity had no significant change (P = 0.1031) (A,C). Data are means ± SEM (n=6-8 animals
per group, 3 section per animal’s brain for immunofluorescence staining). * P < 0.05, ** P < 0.01
compared with sham group. Images were acquired with 4X objective. Scale bar = 500µm.
42
2.5.6. ERK pathway-dependent decrease in cell survival contributes to memory impairment
in SCCH mice
We next investigated whether SCCH induced potential neuronal death and concomitant structural
changes in the hippocampus. In addition, we investigated the effects of SCHH on the activity of
ERK pathway and mitogen-activated protein kinase (MAPK) pathway, which are involved in cell
survival (138,139,296,297), apoptosis (152) and inflammation (153,298). FJB staining was used to
observe neuronal death, whereas Nissl staining allowed visualizing structural changes in
hippocampal regions. Stroke animals were used as a control for cellular death (Supplementary Fig.
Figure 2.6. Low glucose environment alters the activity and the phagocytosis capacity of microglia.
Viability (A), global activity, microglial activation and phagocytosis capacity are studied respectively by
XTT assay, Griess assay (B), FASC(C) and phagocytosis assay analysed by FACS (D). Low glucose
environment do not alter cell viability after 24 hours (A). Cells were exposed 24 hours to 750 µM of H2O2
for death control (A). Low glucose medium significantly decrease global activity (B), microglial
activation (C) and phagocytosis capacity (D). Negative control for phagocytosis assay was performed on
ice (D). DMEM High and DMEM Low respectively represent cells exposed 24 hours to DMEM with
4500mg/L glucose and DMEM with 1000mg/L glucose. Data are means ± S.D. (n=5 experiments per
condition, 4 replicates per experiment for viability assay; n = 6 experiments per condition, 3 replicates per
experiment for Griess assay; n = 5-6 samples per condition for flow cytometry). * P < 0.05, ** P < 0.01,
*** P < 0.005, **** P < 0.0001 compared with DMEM High group; #### P < 0.0001 compared with
control.
43
2.4A) . Neither sham nor SCCH induced detectable neuronal death (Supplementary Fig. 2.4A).
However, structural analysis of the hippocampus revealed that the area of the CA3 region slightly
deceased (P=0.0739) in SCCH mice compared to sham mice (Supplementary Fig. 2.4B). The area
comprising the CA1-2 region and dentate gyrus remained similar for both groups (Supplementary
Fig. 2.4B) . This tendency towards an atrophy of the CA3 region might be linked to a disruption of
cell survival pathways such as MAPK signaling. The stress-activated protein kinases (SAPK)/Jun
amino-terminal kinases (JNK) and p38 MAPK pathways remained similar in both groups for the
duration of the experiment (data not shown). Interestingly, a highly significant (P=0.0161) decrease
in ERK1/2 phosphorylation was observed in SCCH mice (Fig. 2.7B) while total ERK1/2 protein
remained constant (Fig. 2.7A). The ERK pathway has been shown to inhibit neuronal apoptosis
(296,297,299) while promoting cell cycle progression (138) and cell proliferation (139,296). These
results suggest that SCCH alters in parallel the activity of pro-survival pathways, thus contributing
to cognitive decline.
2.6. Discussion
Investigating the impact of chronic hypoperfusion on the progression of AD could lead to a better
understanding of disease’s etiology. Using new model of severe chronic cerebral hypoperfusion (i.e
SCCH) in APPswe/PS1 mice, we found that chronic hypoperfusion affects microglial activation and
Figure 2.7. SCCH lowers ERK1/2 activation. Using Western blot analysis, ERK1/2 total (A) and
phosphorylated protein (B) were quantified and rationalised by β-actin’s level of expression. SCCH had
lower phosphorylation rate of ERK1/2 compared to sham (B) without any change of the total protein
expression (A). Data are means ± SEM (n=5-7 animals per group). * P < 0.050 compared with sham.
44
phagocytosis, which was associated with an increased number of Aβ plaques and a significant
decline in cognitive function.
Here, we report that cerebral hypoperfusion exacerbates the decline in spatial learning as well as in
recognition memory observed of mice subjected to SCCH. Importantly, both sham and SCCH mice
did not exhibit any motor impairment following surgery. Although chronic hypoperfusion has been
correlated to cognitive decline (292), only few studies have examined this correlation in AD
experimental models. For instance, mild chromic cerebral hypoperfusion induced by one-vessel
occlusion (1VO) has been shown to exacerbate memory deficit in transgenic mice overexpressing
human APPswe(126,137). Studies using the BCAS model showed learning impairment in J20/APP
(139). By suing a new model of severe chronic cerebral hypoperfusion, our results are in line with
these previous reports, confirming the pathological association between CBF reduction and
cognitive decline. Our study shows that the detrimental effects of SCCH are not associated to BBB
breakdown or neuronal death, suggesting that the cognitive impairment is possibly associated to
exacerbated neuronal dysfunction. Accordingly, it has been shown that oligemia associated to
cerebral hypoperfusion triggers neuronal dysfunction without necessary inducing neuronal death
(121,126). In contrast with ischemia (139,149,151–155), SCCH model did not induce white matter
injury. Taken together, the latter observations suggest that SCCH affects brain homeostasis (i.e.
severe oligemia) leading to the exacerbation of AD-like pathology in APPswe/PS1 mice.
It has been reported that hypoperfusion affects Aβ processing. In our study, SCCH increased the
number of Aβ plaques in APPswe/PS1 mice without inducing significant changes in Aβ plaque size
and soluble Aβ levels. Consistent with our findings, previous studies have reported alterations in Aβ
processing and deposition in oligemic conditions. More precisely, upon sever oligemia induced by
hypoxia, Koike et al (2010) reported an enhancement of β-secretase protein expression, which
participates in Aβ processing and increases toxic soluble Aβ (140). In contrast with this hypoxic
model (140), our results did not show changes in soluble Aβ load. On the other hand, our data
suggest that Aβ deposits contribute to memory loss, which is in line with a previous study from our
groups, in which Pimentel et al (2013) found a correlation between the cognitive decline and Aβ
plaque number (126). Similar results were also obtained after stenosis in Tg-SwDI mice (144). Both
studies also attributed cognitive decline to significant increase in Aβ plaques.
Cerebral hypoperfusion also triggers vascular stress (300). Here, we report that SCCH induces a
slight increase in the frequency of circulating Ly6CLow, which might be caused by vascular stress.
Following ischemia, an increase in monocyte recruitment has been correlated with vascular
45
remodeling (300) which promotes neuronal survival (301). Moreover, arteriogenesis correlates with
an alternative activation of monocytes (302). In ischemia, Ly6CHigh monocytes are recruited to
phagocyte debris due to acute inflammation (294,295,303), whereas Ly6CLow recruitment occurs
later to promote survival during prolonged injury (299). In the late phase, i.e. five days following
injury, the predominance of patrolling monocytes has been associated with healing, which involves
myofibroblast accumulation, angiogenesis and collagen deposition (295). In our study, although the
increase in patrolling monocytes is not slight, we nonetheless observed a strong tendency at the
10% level 15 weeks after SCCH induction. According to earlier investigations, the increase in
Ly6CLow abundance might be caused by undetectable molecular events that promote vascular
remodeling such as arteriogenesis, which is known to occur during the hypoperfusion-induced
compensation (302).
As mentioned, the efficacy of microglia in eliminating Aβ decreases over time in AD, which have
ben linked to the formation of a stressful environment that alters microglial function (284). These
results outline the impact of the microenvironment on microglial cell function. Here, we report that
SCCH deeply affected microglial cell function, by reducing their activation and recruitment toward
Aβ plaques, which might account for the higher number of plaques observed here. Microglial
glucose demand and consumption increase during activation and phagocytosis since glucose is
required for adenosine triphosphate (ATP) production (304). Moreover, ATP is essential to sustain
phagocytosis (304). Therefore, a glucose-poor environment cannot support the metabolic needs
necessary for microglial activation. Consequently, Aβ clearance through microglia is disrupted,
leading to amyloid deposition. As glucose is the main fuel for brain cells (305), deficiency in this
essential substrate resulting from cerebral hypoperfusion could also disturb the activity of
microglia. To address this point, we used an in vitro approach demonstrating that the low-glucose
microenvironment that is thought to result from the chronic cerebral hypoperfusion, significantly
decreased the metabolic activity of microglia, thereby decreasing microglial activation and
phagocytic capacity.
In addition, we observed a global reduction in ERK1/2 pathway activation as a result of SCCH.
Following phosphorylation ERK1/2 pathway is activated, consequently mediating important roles
in cell survival (300,302,306,307), apoptosis (297,301) and inflammation (295,296). ERK1/2
pathway activation is implicated in the neuroprotective effects of several growth factors, including
insulin-like growth factor 1 (IGF1) and brain-derived neurotrophic factor (BDNF) (297,300,307).
The pathway is also involved in vascular remodeling (308), neurite growth (306) and reactive
oxygen species suppression (303). As such, it is conceivable to speculate that ERK1/2 pathway
46
deactivation following SCCH may be implicated in exacerbating neuronal dysfunction in the brain
of APPswe/PS1 mice, thus possibly contributing to the cognitive decline.
Taken together, our study unravels unknown mechanisms via which chronic cerebral hypoperfusion
affect AD pathogenesis. Importantly, we highlighted the implication of glucose metabolism
impairment in microglial dysfunction, which contributes to the exacerbation of the
neurodegenerative processes. As such, therapeutic strategies aiming to modulate microglial cell
activity should consider the impact of glucose metabolism.
2.8. Acknowledgements
We thank Mohammed Filali, Marie-Michèle Plante, Paul Préfontaine and Nataly Laflamme for their
technical assistance. We also thank Mr. Richard Poulin who contributed in editing the manuscript of
this article.
2.9. Grant support
This work was supported by the Canadian Institutes for Health Research (CIHR).
47
Chapitre 3
3. Tissue-plasminogen activator attenuates Alzheimer’s disease-related
pathology development in APPswe/PS1 mice
Ayman ElAli, PhD, Maude Bordeleau, BSc, Peter Thériault, MSc, Mohammed Filali, PhD, Antoine
Lampron, PhD, and Serge Rivest, PhD
Correspondence:
Dr. Serge Rivest
Neuroscience Laboratory
CHU de Québec Research Center (CHUL)
Department of Molecular Medicine, Faculty of Medicine, Laval University
2705 Laurier boulevard, Québec City
QC G1V 4G2, Canada.
Email: [email protected]
48
3.1. Résumé
La maladie d’Alzheimer (AD) est la première cause de démence chez les personnes âgées. Celle-ci
est caractérisée par l’accumulation du peptide bêta-amyloïde (Aβ) qui s’agrège au fil du temps pour
former des plaques au cerveau. La réduction du niveau d’amyloïde est alors une voie thérapeutique
intéressante. Le cerveau possède plusieurs mécanismes afin de contrôler le niveau de l’Aβ cérébrale
parmi lesquels figurent le système de l’activateur tissulaire du plasminogène (t-PA) / plasmine et les
microglies. Toutefois, ces mécanismes sont défaillants et ineffectifs dans la AD. Dans la présente
étude, nous démontrons que l’administration systémique du t-PA recombinant (Activase® rt-PA)
atténue la pathologie associée à la AD chez des souris transgéniques APPswe/PS1 en réduisant le
niveau d’Aβ cérébrale et en améliorant les fonctions cognitives. Il s’agit d’effets indépendants de
l’activité protéase de rt-PA. Nous observons que rt-PA induit essentiellement une légère
augmentation transitoire de la fréquence des monocytes patrouilleurs dans la circulation en plus
d’une stimulation soutenue des microglies vers un phénotype neuroprotecteur contribuant à
l’élimination de l’Aβ. Notre étude dévoile un rôle pour t-PA dans la maintenance de la capacité de
phagocytose des microglies sans exacerber la réponse inflammatoire. Donc, t-PA constitue une
approche intéressante pour stimuler la population des cellules éliminant de l’Aβ du cerveau.
3.2. Abstract
Alzheimer's disease (AD) is the leading cause of dementia among elderly population. AD is
characterized by the accumulation of beta-amyloid (Aβ) peptides, which aggregate over time to
form amyloid plaques in the brain. Reducing soluble Aβ levels and consequently amyloid plaques
constitute an attractive therapeutic avenue to, at least, stabilize AD pathogenesis. The brain
possesses several mechanisms involved in controlling cerebral Aβ levels, among which are the
tissue-plasminogen activator (t-PA)/plasmin system and microglia. However, these mechanisms are
impaired and ineffective in AD. Here we show that systemic administration of recombinant t-PA
(Activase® rt-PA) attenuates AD-related pathology in APPswe/PS1 transgenic mice by reducing
cerebral Aβ levels and improving cognitive function. Interestingly, these effects were rt-PA
protease activity independent. We observed that rt-PA essentially mediated a slight transient
increase in the frequency of patrolling monocytes together with a sustained stimulation of resident
microglia towards a neuroprotective phenotype, both of which contribute to Aβ elimination. Our
study unraveled a new role of t-PA in maintaining the phagocytic capacity of microglia without
exacerbating the inflammatory response and therefore constitutes an interesting approach to
stimulate the key populations of cells involved in Aβ clearance from the brain.
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3.3. Introduction
Amyloid deposits and neurofibrillary tangle formation are the core pathological hallmarks of AD
(309). Clinically, AD manifests with early mild memory deficits that evolve with time to reach
severe cognitive impairment and consequently the loss of executive functions (310). Despite all
efforts, no efficient treatment exists for AD. However, strategies targeting amyloid deposition seem
promising.
The brain possesses several sophisticated mechanisms that tightly control Aβ processing and
clearance. Importantly, BBB dysfunction has been reported at the early stages of AD pathogenesis
(42), which impairs brain microenvironment, thus deeply impacting brain resident microglia
activity (42). Activated microglia adopt diverse phenotypes ranging from a “classical activation”
(i.e. pro-inflammatory) phenotype that exacerbates inflammation, to an “alternative activation” (i.e.
anti-inflammatory) phenotype that helps in tissue repair (311,312). Microglia have been
demonstrated to contribute to Aβ clearance (47). However, Aβ clearance by resident microglial
cells is extremely slow and ineffective in AD brain (68). Besides microglia, monocytes play
important roles in AD (284,313). Monocytes are mononuclear phagocytic cells and constitute a
population of circulating leukocytes that are central cells of the innate immune system (23). In
rodents, monocytes are regrouped into two main subsets based on chemokine receptors and Ly6C
expression levels, the pro-inflammatory subset (CX3CR1LowCCR2+Ly6CHigh), which is actively
recruited to inflamed tissues and contributes in inflammatory responses, and the anti-inflammatory
subset (CX3CR1HighCCR2-Ly6CLow) that constitutes the resident patrolling monocyte population,
which patrols the lumen of blood vessel and promote tissue repair (23). Our group has recently
demonstrated that the patrolling anti-inflammatory monocyte subset, have the capacity to eliminate
Aβ within the brain vasculature, thus reducing overall cerebral Aβ levels (284). Importantly, the
expansion and the phagocytic capacity of monocytes decrease with age and in AD patients (23).
T-PA is a serine protease that converts plasminogen into plasmin, an enzyme involved in fibrin
degradation, which has been reported to be also involved in Aβ microaggregate degradation (162).
In parallel, t-PA has been proposed to act as an anti-inflammatory cytokine, independently from its
protease activity (197). Interestingly, similar to microglia spatial localization, t-PA expression and
activity have been reported to localize around Aβ plaques in the brain of a mouse model of AD,
suggesting its implication in Aβ processing (162). Unfortunately, over time, the t-PA/plasmin
system gets inefficacious in degrading Aβ microaggregates (162).
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In the present study, we investigated the therapeutic potential of Activase®, a rt-PA that is approved
by the Food and Drug Administration (FDA) for ischemic stroke treatment, in modulating AD
pathology. For this purpose, we used four months old APPswe/PS1 mice that were intravenously
treated with low doses of Activase® r-tPA (5mg/kg/week for 10 weeks). Here we report that rt-PA
significantly delayed the progression of AD pathology, which was mediated by its anti-
inflammatory characteristics. More precisely, rt-PA reduced soluble Aβ levels and Aβ plaque
number and size and improved cognitive function without affecting BBB function and integrity.
Interestingly, rt-PA treatment enhanced the coverage of Aβ plaques by microglia and triggered their
“alternative activation” via LRP1. Moreover, rt-PA specifically increased the subset of patrolling
monocytes, which are involved in the clearance of vascular Aβ. Taken together, our study
demonstrates that rt-PA may constitute a novel approach to treat AD by enhancing the anti-
inflammatory reparative phenotype of microglia and monocytes.
3.4. Materials and Methods
3.4.1. Animal experiments
Experiments were performed according to the Canadian Council on Animal Care guidelines, as
administered by the Laval University Animal Welfare Committee. All efforts were made to reduce
the number of animals used and to avoid their suffering. Four month old APPswe/PS1 transgenic
mice harboring the human presenilin I (A246E variant) and the chimeric mouse/human Aβ
precursor protein (APPswe) under the control of independent mouse PrP promoter elements [B6C3-
Tg(APP695)3Dbo Tg(PSEN1)5Dbo/J] (Jackson ImmunoResearch Laboratories Inc., West Grove,
PA, USA) maintained in a C57BL/6J background. C57BL6/J mice (wildtype) littermates were used
as controls. Additional set of green fluorescent protein (GFP)+/- mice were used to generate chimeric
mice. Mice were housed and acclimated to standard laboratory conditions (12-hour light/dark cycle
/ lights on at 7:00 AM and off at 7:00 PM) with free access to chow and water. Only males were
used at the age of 4 months. Four months old mice were used as around this stage the plaques begin
to develop in the mouse line used in the laboratory. APPswe/PS1 mice were treated intravenously
with a single dose of 5 mg/kg per week of Activase® rt-PA (Roche, Mississauga, ON, Canada) over
a total period of ten weeks. As rt-PA has a short half-life in circulation and could trigger BBB
breakdown following administration in stroke, a regimen of chronic low doses that represent half
the dose usually used for ischemic stroke thrombolysis, has been chosen. Twenty-four hours after
last rt-PA injection, blood was collected from awaken mice via the facial vein for further analysis.
Additional group of APPswe/PS1 mice were used to collect blood 3 hours following rt-PA injection
for further analysis. The week that followed blood collection, behavioral analysis was performed,
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mice were then sacrificed and tissues were collected as described here below. In another set of
experiments, wildtype mice were treated intravenously with a single 5 mg/kg dose of Activase® rt-
PA, 3 and 24 hours after injection blood was collected from awaken mice via the facial vein for
further analysis, mice were then sacrificed and tissues were collected as described here below. A
group of APPswe/PS1 chimeric mice, in which the original bone marrow-derived cells were
replaced by bone marrow-derived cells of GFP+/- mice, were generated by a myeloablative
chemotherapy regimen that preserves the BBB, which were treated with Activase® rt-PA and killed
24 hours following injection. Another group of APPswe/PS1 chimeric mice were generated by
total-body irradiation that alters the BBB, which were used as positive controls for GFP-positive
blood-borne cell infiltration into the brain. Brain tissues were collected as described here below.
Finally, two groups of APPswe/PS1 mice were treated with a single 5mg/kg dose of Activase® rt-
PA, 3 and 24 hours following injection mice were sacrified and brains removed and directly
processed to isolate microglia for flow cytometry analysis.
3.4.2. Chimeric mice generation
APPswe/PS1 chimeric mice were generated by transplanting bone marrow-derived cells of GFP+/-
mice in myeloablated APPswe/PS mice, as described previously (314). Briefly, APPswe/PS1
recipient mice were given water containing a commercially available mix of antibiotics (SEPTRA;
GlaxoSmith Kline, Mississauga, Ontario, Canada) for 1 week before starting a myeloablative
chemotherapy regimen consisting of twice-daily injections (morning and evening) of 10 mg/kg
busulfan for 4 days (a total of 80 mg/kg), followed by daily injections of 100 mg/kg
cyclophosphamide for 2 days (a total of 200 mg/kg). The injections were performed in a total
volume of 150 μl intraperitoneally, alternating sides between each injection. To counter
chemotherapy-induced dehydration, mice received a daily 1 ml injection of saline subcutaneously
for 1 week. Mice were then transferred to sterile cages and given previously irradiated food.
Antibiotic treatment continued for 1 week following treatment. On the day following the last
injection of cyclophosphamide, GFP+/− donor mice were killed by cervical dislocation with
isoflurane anesthesia. Their femurs and tibias were dissected, and their bone marrow was flushed
with phosphate-buffered saline (PBS) containing 5% FBS (Sigma-Aldrich, St. Louis, MO, USA).
The cells were filtered through 35-μm nylon mesh (BD Bioscience, San Jose, CA, USA), washed
three times in FBS-free PBS (centrifuging at 300g for 5 minutes between washes), and counted with
a hematocytometer. Cells (1.5 × 107) were then injected into the tail vein of recipient mice. They
were followed for 4-6 weeks before they received any other treatment or surgeries to allow the
injected cells to repopulate the hematopoietic system. Another group of APPswe/PS1 chimeric mice
52
were generated by exposition to a 10 gray total-body irradiation using a cobalt-60 source
(Theratron-780 model, MDS Nordion, Ottawa, ON, Canada), instead of chemotherapy.
3.4.3. Tissue collection
For molecular analysis, mice were deeply anesthetized via an intraperitoneal injection of a mixture
of ketamine hydrochloride/ xylazine (100/10 mg/kg) and then transcardially perfused with ice-cold
0.9% saline solution (0.9% NaCl) (Sigma-Aldrich) by using a peristaltic pump, brains were
removed and immediately processed for microglia islation or frozen in dry ice for subsequent
molecular analysis. For immunofluorescence and histochemical analysis, mice were anesthetized as
above and then transcardially perfused with ice-cold 0.9% saline solution, followed by 4%
paraformaldehyde (PFA, Sigma-Aldrich) in 0.1 M PBS, brains were removed and postfixed in 4%
PFA (pH 7.4) at 4 °C and then immersed in a PFA solution containing 20% sucrose overnight at 4
°C. Fixed brains were frozen with dry ice/ethanol mixture, mounted on a microtome (Leica,
Concord, ON, Canada) and cut into 25 μm coronal sections. The collected slices were placed in
tissue cryoprotectant solution containing 0.05 M sodium phosphate buffer (pH 7.3), 30% ethylene
glycol, and 20% glycerol, and stored at −20 °C until analysis. Blood samples were collected in
ethylenediaminetetraacetic acid (EDTA) coated tubes during the protocol via the facial veins, and
mice were allowed to rest one week after bleeding.
3.4.4. Immunofluorescence staining
Free-floating sections were washed with KPBS (Sigma-Aldrich) (3x, 10 minutes) and then
incubated for 20 minutes in a permeabilization/blocking solution containing 4% goat serum, 1%
bovine serum albumin (BSA) (Sigma-Aldrich), and 0.4% Triton X-100 (Sigma-Aldrich) in KPBS.
Sections were incubated overnight at 4 °C with different primary antibodies diluted in the same
permeabilization/blocking solution. The following primary antibodies were used; mouse anti-human
Aβ monoclonal antibody (6E10) (1/1500) (SIG-39320, Covance Inc., Princeton, NJ, USA), rabbit
anti-Iba1 (1/1500) (019-19741; Wako Chemicals, Richmond, VA, USA), rat anti-mouse CD31
antibody (BD Bioscience). Afterwards, the sections were rinsed in KPBS (3x, 10 minutes),
followed by a 2 hours incubation with either Cy3-conjugated goat anti-mouse secondary antibody
(115-165-003; Jackson ImmunoResearch Laboratories, West Grove, PA, USA), or Alexa Fluor
488-conjugated goat anti-rabbit secondary antibody (A11008; Life Technologies Inc., Burlington,
ON, Canada). Sections were incubated overnight under light protected vacuum to allow an optimal
fixation of brain sections on slides. The next day, sections were rinsed in KPBS (3x, 10 minutes),
stained with 0.0002% DAPI for 5 minutes, rinsed again in KPBS (3x, 10 minutes), mounted onto
SuperFrost slides (Fisher Scientific, Ottawa, ON, Canada), and coverslipped with antifade medium
53
composed of 96 mM Tris-HCl, pH 8.0, 24% glycerol, 9.6% polyvinyl alcohol, and 2.5%
diazabicyclooctane (Sigma-Aldrich). Epifluorescence images were taken using a Nikon C80i
microscope equipped with both a motorized stage (Ludl, Hawthorne, NY, USA) and a Microfire
CCD color camera (Optronics, Goleta, CA, USA). Confocal laser scanning microscopy was
performed with a BX-61 microscope equipped with the Fluoview SV500 imaging software 4.3
(Olympus America Inc., Melville, NY, USA).
3.4.5. IgG and albumin extravasation
Free-floating sections were washed with KPBS (3x, 10 minutes) and then incubated for 20 minutes
in the permeabilization/blocking solution containing 4% goat serum, 1% bovine serum albumin
(BSA) (Sigma-Aldrich), and 0.4% Triton X-100 (Sigma-Aldrich) in KPBS. For IgG detection,
sections were incubated for 2 hours with biotin-conjugated goat anti-mouse secondary antibody
(1/1000) (BA9200; Vector Laboratories, Burlingame, CA, USA). For albumin detection, sections
were incubated overnight at 4 °C with anti-mouse serum albumin (1/1000) (ab19194; Abcam Inc.,
Toronto, ON, Canada) diluted in the same permeabilization/blocking solution. The Biotin-
conjugated secondary antibodies were detected using the avidin peroxidase kit (Vectastain ABC kit,
Vector Laboratories) and diaminobenzidine (Sigma-Aldrich), following the manufacturer's
instructions. Sections were then mounted onto SuperFrost slides (Fisher Scientific), dehydrated and
coverslipped with DPX mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA).
Bright light images were taken using the Nikon C80i microscope equipped with the motorized stage
(Ludl) and a Microfire CCD color camera (Optronics).
3.4.6. Aβ plaques, microglia coverage and Aβ internalization by microglia quantification
Aβ plaques were stained with an anti-human Aβ monoclonal antibody (6E10) as described
previously (313). Aβ plaque number and size were assessed in the hippocampus and the overlaying
cortex separately using a stereological apparatus as described (315). Briefly, real-time images (1600
× 1200 pixels) were obtained using the Nikon C80i microscope equipped with the motorized stage
(Ludl) and a Microfire CCD color camera (Optronics). Both cortex and hippocampus areas were
traced using a Cintiq 18S interactive pen display (Wacom, Ontone, Saitama, Japan). The contours
of the cortex or hippocampus areas were traced as virtual overlay on the steamed images. Aβ plaque
number and area occupied by Aβ immunostained plaques within these traced virtual regions were
determined. In order to assess microglia coverage of Aβ plaques, immunostained brain sections
(sections through the hippocampus region) were analysed using the stereological apparatus as
described previously (315). Briefly, four brain sections per animal stained for microglia (Iba1), Aβ
(6E10) and nuclei (DAPI) were blindly assessed. Aβ plaques were traced and microglia counted for
54
each frame using the pen display, a 40× Plan Apochromat objective (NA 0.95). In order to
investigate Aβ internalization by microglia in vivo following rt-PA chronic treatment, Aβ-
immunoreative microglia were assessed by quantifying Aβ positive immunosignals in microglial
cell body. These methods generate semi-quantitative data highly representative of the general state
of the animal (313). Moreover, this method was designed to ensure that this type of quantification
was representative of the total amount of plaques and microglia determined by unbiased
stereological quantification (313). Additional brain sections were double stained for CD45
(infiltrating leukocytes) and Iba1 (differentiated microglia) in order to investigate the infiltration of
blood-derived monocytes into the brain and their differentiation into macrophages.
3.4.7. In situ Hybridization
Brain sections were mounted on Colorfrost/Plus microscope slides (Fisher Scientific). In situ
hybridization histochemical localization of inhibitor of kappa B alpha (IκBα), which is used as an
index of nuclear factor-kappa B (NF-kB) activity was performed using 35S-labeled cRNA probes.
Plasmids were linearized and sense/ antisense cRNA probes were synthesized with an appropriate
RNA polymerase. All plasmids were analysed for sequence confirmation and orientation.
Riboprobe synthesis and preparation as well as in situ hybridization were performed according to a
protocol described previously (316). All slides were developed on the same films that were scanned
and densitometrically analysed using ImageJ image analysis software (NIH).
3.4.8. Soluble Aβ1–42 Enzyme-Linked Immunosorbent Assay (ELISA)
Brain levels of soluble Aβ1–42 were quantified using the Human Amyloid β42 Brain ELISA kit
(Millipore, Billerica, MA, USA). The experimental procedure for Aβ1–42 detection was performed
according to the manufacturer's instructions. Briefly, brains were homogenized in ice cold lysis
buffer, and centrifuged at 2500xg for 10 minutes at 4 °C. Supernatant was diluted and loaded into
96 wells microplate. Absorbance was acquired using a microtiter plate reader (SpectraMax 340PC,
Molecular Devices, San Diego, CA, USA), and analysed using SOFTmax Pro3.1.1 software
(Molecular Devices).
3.4.9. Brain microvessel isolation
Brain capillaries were isolated on dextran gradient as described previously (317), with slight
modification. Briefly, the cerebellum, meninges, brainstem and large superficial blood vessels were
removed and the remaining cortices were gently homogenized in a Teflon glass homogenizer in ice-
cold microvessel (MVs) (i.e. capillaries) isolation buffer (MIB; 15 mM HEPES, 147 mM NaCl, 4
mM KCl, 3 mM CaCl2, and 12 mM MgCl2) supplemented with 5% Protease Inhibitor Cocktail
55
(P8340; Sigma-Aldrich) and 1% Phosphatase Inhibitor Cocktail 2 (P5726; Sigma-Aldrich).
Homogenates were centrifuged at 1000xg for 10 min at 4°C. The resulting pellets were resuspended
in 30% dextran (molecular weight, 64,000 to 76,000; D4751, Sigma) in MIB. Suspensions were
centrifuged at 4400xg for 20 min at 4°C. The resulting crude brain capillaries-rich pellets were
resuspended in MIB and filtered through two nylon filters of 100- and 60-μm mesh size (Millipore).
The quality of trapped brain in the final filtrates was checked with a bright-field microscopy
(Supplementary Fig. 1a). Filtrates were centrifuged at 1000xg, and the resulting pellets that
contain the pure brain capillaries were stored at −80°C until further use.
3.4.10. Microglia’s isolation and analysis by flow cytometry
Mice were sacrified as described previously. Brains were removed and homogenized in 1 ml ice
cold Dulbecco's PBS (DPBS) without calcium (Ca²+)/ magnesium (Mg²+). Homogenized brain
samples were washed for 10 minutes at 300xg at 4°C, and resuspended in 3 ml digestion buffer
containing a cocktail of enzymes, TLCK (0,5 µg/ml) (Sigma), DNAse1 (25 ng/ml) (Roche),
Liberase (8,125pg/ml) (Roche), HEPES 20mM (Sigma) in 0.1 M DPBS, and incubated for 40
minutes at 37°C. Afterwards, the volume was completed to 10 ml by adding 7 ml of flow cytometry
buffer containing 5% FBS, EDTA (20 mM), HEPES (2 mM) in flow cytometry buffer. The digested
samples were filtered through a 70 µm sterile nylon filter, washed once with flow cytometry buffer,
and centrifuged for 10 minutes at 300xg at 4°C. The resulting pellet was raised in 8 ml 30% Percoll
(GE Healthcare Life Sciences, Baie d’Urfe, QC, Canada), which was diluted in a solution
containing EDTA (2 mM), HEPES (20 mM), phenol red, 1X Hank's balanced salt solution (HBSS).
4 ml of the 30% Percoll containing samples were loading carefully at the bottom of a 15 ml falcon
tube containing 4 ml 80% Percoll solution, which was diluted in a solution containing EDTA (2
mM), HEPES (20 mM) (Sigma) and 1X HBSS. The remaining 4 ml of the 30% Percoll containing
samples were carefully added at the surface of the 80% Percoll solution. Afterwards, the gradient
column was centrifuged for 40 minutes at 1000xg at 18°C with low acceleration and no brake.
Microglial cell enriched fractions were collected from the inter-phase, resuspended in 10 ml of flow
cytometry buffer, and centrifuged for 10 minutes at 300xg. The resulting pellet, which contains the
pure fraction of microglia, was resuspended in 250 µl of flow cytometry buffer, and transferred to a
96-wells conical plate. Cells were incubated for 15 min on ice with rat anti-mouse CD16/CD32
antibody (Mouse BD Fc Block; BD Bioscience) diluted in flow cytometry buffer. Afterwards, cells
were centrifuged for 3 min at 300xg at 4°C, and incubated for 30 minutes with PE (phycoerythrin)-
Cy5-conjugated anti-CD45 antibody (BD Bioscience), Alexa Fluor® 700-conjugated anti-CD11b
antibody (eBioscience, San Diego, CA, USA), and LIVE/DEAD® blue fluorescent dye (Life
56
technologies). Cells were incubated with the antibody mixture for 30 minutes at 4°C. Cells were
washed for 10 minutes at 300xg with DPBS without Ca²+/Mg2+, and resuspended in flow cytometry
buffer. Finally, the cells were analysed using a LSR II flow cytometer and data acquisition was
done with BD FACS Diva software (Version 6.1.2, BD Bioscience). The results were analysed
using FlowJo software (Version 7.6.1, Tree Star Inc., Ashland, OR, USA).
3.4.11. Protein extraction
Isolated brain capillaries or brain tissues were homogenized and lysed in NP-40 lysis buffer
supplemented with 5% Protease Inhibitor Cocktail and 1% Phosphatase Inhibitor Cocktail 2
(Sigma-Aldrich). Lysate samples were sonicated over two cycles lasting 20 s each at 4°C at 40%
power. Protein concentrations were measured by means of the Quantipro BCA assay kit (Sigma-
Aldrich) according to the manufacturer’s protocol. Absorbance was acquired using a microtiter
plate reader (SpectraMax 340PC, Molecular Devices), and analysed using SOFTmax Pro3.1.1
software (Molecular Devices).
3.4.12. Caseinase and gelatinase activity assays
In order to investigate the enzymatic activity of plasmin (caseinase), we used a highly sensitive
fluorescent based assay “Sensolyte® AFC Plasmin Activity Assay Kit” (AnaSpec, Fremont, CA,
USA). In order to investigate the enzymatic activity of MMP-2/9 (gelatinase) we used a highly
sensitive fluorescent based assay “EnzCheck® Gelatinase/ Collagenase Assay Kit” (Molecular
Probes, Eugene, OR, USA). The caseinase and gelatinase assays were performed according to
manufacturer’s protocols, respectively.
3.4.13. Western blot analysis
For total and phosphorylated protein analyses, 2X SDS loading buffer was added to lysate samples
containing equal amounts of protein (10 µg). These samples were heated for all protein analysis
studies except for those involving ABCB1, for which samples were loaded without heating to avoid
aggregation of these highly glycosylated transmembrane proteins. Samples were subjected to SDS–
polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis, with primary
antibodies diluted 1:1000 in 5% skim milk (Sigma-Aldrich) and 0.1 M tris-buffered saline–Triton
X-100 (TBS-T). The following antibodies were used: Rabbit anti-ABCB1 (sc-8313) and rabbit anti-
claudin 5 (sc-28670) were purchased from Santa Cruz Biotechnology, Dallas, TX, USA. Rabbit
anti-total SAPK/JNK (9252), anti-phopho SAPK/JNK (9251), anti-total p38 MAPK(9212) and anti-
phospho-p38 MAPK (9211) were purchased from Cell Signaling Technology, Danvers, MA USA.
Rabbit anti-LRP1 (ab92544) and anti-RAGE (ab37647) were obtained from Abcam Inc. Rabbit
57
anti-occludin (71-1500) was purchased from Life Technologies Inc., and anti-β-actin (MAB1501)
was purchased from EMD Millipore, Billerica, MA, USA. Primary antibodies were detected with
HRP–conjugated secondary IgG that were diluted 1:5000 in 5% skim milk and TBS-T and revealed
by ECL solution (GE Healthcare Life Sciences). Blots were digitized, densitometrically analysed
with ImageJ image analysis software (NIH), corrected for protein loading by means of β-actin, and
expressed as relative values comparing control groups with treated groups.
3.4.14. Flow cytometry
Flow cytometry -analysis was used to determine the population of monocytes in the circulation.
Facial vein blood was collected in EDTA coated vials (Sarstedt, Newton, NC, USA). Flow
cytometry analysis was performed as described (314). Briefly, 50 µl of total blood was incubated on
ice for 15 minutes with 4 µl purified rat anti-mouse CD16/CD32 antibody (BD Bioscience) diluted
in 35 µl DPBS. Always on ice, the mixture of cells and anti-mouse CD16/CD32 was incubated with
PE-Cy7-conjugated anti-CD11b antibody (eBioscience), APC (allophycocyanin)-conjugated anti-
CD115 antibody (eBioscience), PE-conjugated anti-CD45 antibody (BD BioScience) and
fluorescein isothiocyanate-conjugated anti-Ly6-C antibody (BD BioScience) for 45 minutes. Red
blood cells were lysed with 1.5 ml Pharm Lyse buffer, according to manufacturer's protocol (BD
BioScience). After hemolysis, remaining cells were washed with DPBS and resuspended in equal
volumes of DPBS. Finally, the cells were analysed using a LSR II flow cytometer and data
acquisition was done with BD FACS Diva software (Version 6.1.2, BD Bioscience). The results
were analysed using FlowJo software (Version 7.6.1, Tree Star Inc.).
3.4.15. Behavior analysis
The T-water maze paradigm was used to assess mice spatial learning and memory, in the weeks
following the last injection (318). This paradigm evaluates the ability of mouse to remember the
spatial location of a submerged platform. The T-maze apparatus (length of stem, 64 cm; length of
arms, 30 cm; width, 12 cm; height of walls, 16 cm) was made of clear fiberglass and filled with
water (23 ± 1 °C) at a height of 12 cm. An escape platform (11 × 11 cm) was placed at the end of
the target arm and was submerged 1 cm below the surface. The position of the platform was chosen
randomly for each animal prior to testing. In the acquisition-learning phase, which allows the
evaluation of left/ right spatial learning, the mice were placed in the stem of the T-maze and swam
freely until they found the submerged platform (located either in the right or in the left arm of the T-
maze apparatus) and escaped to it. After reaching the platform, the mice remained on it for 20
seconds and then placed back in the maze for up to a maximum of 24 trials, except for a 10 minutes
rest period after each 10 trial block. If the animals did not find the platform within 60 seconds, they
58
were gently guided onto it. During the rest period, mice were dried with towels and provided with
heating pads to prevent hypothermia. All trials were performed on one single day. A mouse was
considered to have achieved criterion after 5 consecutive errorless trials. The reversal-learning
phase was then conducted 2 days later, with the protocol repeated except that the mice were trained
to find the escape platform on the opposite side. During the acquisition/reversal phase the platform
was located in the same position for the entire stage. The number of trials to reach the criterion (5 of
5 correct choices made on consecutive trials) and the average of swimming speeds were recorded
and analysed.
Based on their scores during the reversal phase, the animals were subdivided in 3 subgroups (no
cognitive deficit = as defined by values below 10 trials; mild cognitive deficit = as defined by
values between 10-16 trials; severe cognitive decline = as defined by values above 16 trials).
3.4.16. In vitro experiments
3.4.16.1. Cells culture
The immortalized murine microglial cell line (BV2) were cultured at 37°C in 5% CO2, 95% air in
DMEM (Life Technologies Inc.) containing 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin and
100 µg/mL streptomycin. In all experiments, cells were grown to 70-90% confluence and subjected
to a maximum of 20 cell passages.
3.4.16.2. Cell stimulation
BV2 (6x105 cells) were stimulated with 0,1 nM rt-PA (Roche), 2 µg/ml LPS or 5 ng/ml mIL-4 for
30 minutes at 37°C. Afterwards, cells were dissociated using a non-enzymatic cell dissociation
buffer (Sigma-Aldrich), washed twice with PBS, and lysed in 1% NP-40 lysis buffer supplemented
with 5% Protease Inhibitor Cocktail and 1% Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich).
Lysates were gently sonicated using the Sonic dismembrator model 100 (Fisher Scientific). Protein
levels were quantified using the Quantipro BCA assay kit (Sigma-Aldrich). Cell lysates were used
to investigate intracellular signaling pathways using Western blot or enzymatic activity using
fluorescent based assay kits.
3.4.16.3. Cell migration assay
BV2 cells were seeded at 4x104 (cells/ well) in a 24-well plate (BD Falcon®, BD, Mississauga, ON,
Canada). Cell growth was stop by changing the media to DMEM / 1% FBS when the density was
about 80 to 100%. Using a sterile tip, a wound was made in the middle of the well, directly
afterwards cells were stimulated with 0,1 nM rt-PA (Roche), 0,1 nM rt-PA (S478A) (Abcam Inc.), 2
µg/ml LPS, 5 ng/ml mIL-4 and 0,1 nM rt-PA + 200 nM recombinant receptor-associated protein
59
(RAP) (LRP1 inhibitor) (EMD Millipore). Pictures were acquired at 0 and 24 hours post-treatment
using Olympus IX81 inverted research confocal microscope (Olympus America, Center Valley, PA,
USA). Acquired images were analysed using ImageJ image analysis software (NIH), where the
number of infiltrated cells into the scratch was assessed.
3.4.16.4. Chemotaxis assay
Transwell polycarbonate 8 µm inserts (Corning, Lowell, MA) were coated with 2 µg/ml laminin
(Sigma-Aldrich) overnight at 4°C. Inserts were equilibrated in DMEM / 1% FBS at least for 1 hour.
BV2 cells were harvested and seeded at 4-5x104 (cells/ well) with or without 200 nM RAP (EMD
Millipore) that were added to the upper chamber. The lower chamber contained DMEM / 1% FBS
with or without 0,1 nM rt-PA (Roche). The microplate was placed 24 hours in a humidified
incubator at 37°C. Cells were dissociated using a non-enzymatic dissociation buffer, collected from
both compartments and counted with a hemacytometer.
3.4.16.5. Phagocytosis assay
BV2 cells 1x105 (cells/ well) in 96-well microplate were stimulated with 0,1 nM rt-PA (Roche), 0,1
nM rt-PA (S478A) (Abcam Inc.), 1 µg/ml LPS or 5 ng/ml mIL-4 for 1 hour at 37°C. Stimulated
cells were incubated with 1mg/ml fluorescein-labeled E. coli beads (Molecular Probes) for 2 hours
at 37°C. Afterwards, cells were incubated with 250 µg/ml blue trypan (Molecular Probes) at room
temperature for 1 minute. Phagocytosis rate was determined by measuring fluorescence emission at
520 nm following an excitation at 480 nm with a fluorescent plate reader SpectraMAX Gemini
(Molecular Devices).
3.4.16.6. Griess Assay
Oxidative stress was quantified by measuring nitrite release in cell culture medium by using the
Griess Assay according to manufacturer’s protocol (Life Technologies Inc.). Briefly, BV2 cells
were incubated for 24 hours with rt-PA (0,1 nM), LPS (1 μg/ml), IL-4 (5 ng/ml) or kept without
stimulation. The cell culture medium was then transferred into a 96-well plate and mixed with 20
μL of Griess reagent and 130 μL of deionized water. In parallel, a standard curve was created
ranging from 1 µM to 100 µM. The samples and standards were incubated for 30 minutes at room
temperature. The nitrite concentrations was determined by measuring the absorbance at 548 nm by
using a microtiter plate reader (SpectraMax 340PC, Molecular Devices), and analysed using
SOFTmax Pro3.1.1 software (Molecular Devices).
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3.4.17. Statistics
Results are expressed as mean ± SEM. For the cellular and molecular analysis, the comparison
between two group data was analysed using standard two-tailed unpaired t-test. For the behavioral
analysis, the intergroup differences were evaluated by ANOVA followed by the Bonferroni’s post
hoc test or Kruskal–Wallis followed by the Mann–Whitney test. The two-sided Chi2-square was
used to compare cognitive performances in the T-maze. A P value < 0.05 was considered
statistically significant. All analyses were performed using GraphPad Prism Version 6 for Windows
(GraphPad Software, San Diego, CA, USA).
3.5. Results
3.5.1. Activase® rt-PA regimen does not affect blood-brain barrier integrity and function
Endogenous t-PA is localized mainly at the abluminal side of the BBB, where it modulates the
permeability of the latter (170) and the microvascular tone (319). Several reports demonstrated that
excess of t-PA at the abuminal side of the BBB and the parenchyma mediates BBB breakdown and
neuronal death by excitotoxicity (189,320). However, in different contexts, other studies showed
that t-PA is implicated in neuronal synaptic plasticity (186) and is neuroprotective (200). We first
tested the effects of Activase® rt-PA regimen on BBB physical integrity in APPswe/PS1 mice. rt-
PA did not compromise BBB tightness after chronic weekly injections of rt-PA for a period of 10
weeks, which was translated by the absence of albumin (Supplementary Fig. 3.1b) and IgG
(Supplementary Fig. 3.1c) extravasation within brain parenchyma. In order to fully address the
effects of rt-PA on the BBB, the expression levels of proteins involved in BBB physical integrity
and function in wildtype mice were investigated 3 and 24 hours after injection. Interestingly, rt-PA
did not change the expression levels of the tight junction protein, Occludin (Supplementary Fig.
3.2a, b) and Claudin 5 (Supplementary Fig. 3.2c, d). In addition, rt-PA did not change the
expression levels of ABCB1 (Supplementary Fig. 3.2e, f), a transporter involved in brain
detoxification (317,321) and possibly involved in Aβ elimination across the BBB (50). In parallel
rt-PA did not change LRP1 protein levels, a t-PA receptor that is also involved in the clearance of
cerebral Aβ across the BBB (54,322) (Supplementary Fig. 3.3a, b), and RAGE that is involved in
peripheral Aβ entry into the brain via the BBB (Supplementary Fig. 3.3c, d) (40). These results
clearly demonstrate that the Activase® rt-PA regimen did not induce BBB breakdown, and did not
change the expression levels of some key proteins involved in Aβ elimination across the BBB.
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Figure. 3.1. Activase® rt-PA administration reduces Aβ aggregates and soluble Aβ1-42 levels in the
brain. Immunofluorescence staining (a-d) and ELISA (e,f) analyses examining the deposition of Aβ
aggregates and Aβ1-42 / Aβ1-40 soluble levels in the brain of APPswe/PS1 mice, 10 weeks after Activase® rt-
PA weekly systemic administration. The 6E10 immunofluorescence staining shows a decrease in Aβ plaque
number in the cortex (a) and the hippocampus (b) of treated animals. Moreover, 6E10 immunofluorescence
staining shows a reduction in Aβ plaque size in the cortex (c) and the hippocampus (d) of treated animals.
Finally, ELISA analysis shows reduced levels of soluble Aβ1-42 (e) and unchanged levels of soluble Aβ1-40
(f). Data are means ± SEM (n = 8-10 animals per group for both experiments, 3 sections representing the
rostral, middle and caudal levels of the hippocampus and overlaying cortex per animal’s brain for
immunofluorescence staining). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared with
saline treated group. Images were acquired with a 4X objective. Scale bar = 100 µm
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3.5.2. Activase® rt-PA slows the progression of AD-like pathology and behavioral deficits in
APPswe/PS1
It has been proposed that the endogenous t-PA system is involved in cerebral Aβ processing.
Therefore, the impact of Activase® rt-PA regimen on plaque number and size was investigated.
Interestingly, rt-PA systemic administration significantly reduced Aβ plaque number and size in the
cortex (Fig. 3.1a, c) and hippocampus (Fig. 3.1b, d). The toxicity of soluble Aβ in the brain of AD
patients (80) and mouse models of AD (79) has been clearly demonstrated. Interestingly, rt-PA
significantly reduced soluble Aβ1-42 levels (Fig. 3.1e) without affecting the levels of soluble Aβ1-40
(Fig. 3.1f). In order to investigate the physiological relevance of rt-PA-induced Aβ clearance on
mice cognition, we used a T-water maze behavioral paradigm that assesses specifically
hippocampus-based spatial learning and memory (Fig. 3.2). No intergroup difference was seen
during the acquisition phase of the water T-maze behavioural analysis (Kruskal-Wallis = 0.78, p
Figure. 3.2. Activase® rt-PA administration improves APPswe/PS1 mice cognitive functions. T-water
maze behavioral test was used to examine spatial learning and memory (a,b) and to classify the cognitive
performance of mice as a function of treatment (c). Activase® rt-PA treatment does not change the number
of trials to reach criterion in the acquisition phase of the test (a), but significantly enhances the cognitive
functions of APPswe/PS1 mice (b) as shown by their lower number of trials to reach the criterion in the
reversal phase of the test. In addition, the cognitive deficit was highly similar between rt-PA-treated
APPswe/PS1 mice and wildtype mice, which was significantly lower than saline-treated APPswe/PS1 mice
(c). Each point represents an animal and the horizontal bars are the mean for each group. MD: Mild
Cognitive Deficit, ND: No Cognitive Deficit, SD: Severe Cognitive Deficit, WT: wildtype. Data are means
± SEM (n = 13-16 animals per group). * P < 0.05, ** P < 0.001 compared with saline treated group (a,b), *
P < 0.05 compared with saline treated group (c).
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0.05). The three groups of mice took a similar number of trials before reaching criterion
performance (Fig. 3.2a). However, during the reversal phase, the analysis of the trials to criterion
revealed a significant effect (Kruskal-Wallis = 9.52, p < 0.01) (Fig. 3.2b). Moreover, the Mann–
Whitney multiples comparisons revealed that rt-PA-treated APPswe/PS1 mice and wildtype mice
have both lower trials to criterion than saline-treated APPswe/PS1 mice (p < 0.05 and p < 0.01,
respectively) (Fig. 3.2b). During the reversal phase, the three groups of mice did not exhibit any
(Fig. 3.2c), the number of mice exhibiting severe cognitive deficit (SD) was lower in rt-PA-treated
APPswe/PS1 mice and wildtype mice compared to saline-treated APPswe/PS1 mice (Chi2 test, p <
0.05) (Fig. 3.2c). In addition, the number of mice exhibiting no deficit (ND) was higher in rt-PA-
treated APPswe/PS1 mice and wildtype mice compared to saline-treated APPswe/PS1 mice (Chi2
test, p < 0.05) (Fig. 3.2c). These results reveal that the rt-PA-treated APPswe/PS1 and wildtype
mice were largely similar in performance and exhibiting similar cognitive profiles.
Figure. 3.3. t-PA-associated perivascular proteases are not induced by Activase® rt-PA regimen.
Caseinase (a,b), gelatinase (c,d) activity assays examining plasmin (caseinase) and MMP2/9 (gelatinase)
enzymatic activities the brain and microvasculature of APPswe/PS1 mice and their wildtype littermates.
Chronic systemic Activase® rt-PA administration does not change plasmin (a) and MMP2/9 (c) activities in
the brain and the microvasculature of APPswe/PS1 treated mice. In addition, the acute systemic Activase®
rt-PA treatment does not change the plasmin (b) and MMP2/9 (d) activities in the brain and the
microvasculature of wildtyp mice. TBH: Total brain homogenates, WT: Wildtype.
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3.5.3. The enzymatic activity is not responsible of rt-PA-induced clearance of Aβ
In order to shed light on the mechanisms that might be involved in the protective effects of rt-PA,
we first investigated the implication of the enzymatic activity of rt-PA. It has been reported that t-
PA is indirectly involved in Aβ processing. More precisely, t-PA converts plasminogen into
plasmin, which is involved in Aβ degradation (162). Moreover, t-PA has the ability to activate
MMP2/9 (213), two enzymes involved in vascular remodeling (323) and Aβ degradation (214,215).
The Activase® rt-PA regimen used in this study did not modulate the basal enzymatic activity of
both plasmin (Fig. 3.3a) and MMP2/9 (Fig. 3.3c) in the brain and the microvasculature of
APPswe/PS1 treated mice. To verify whether these unexpected results were due to the chronic
systemic rt-PA administration in APPswe/PS1 mice and/or the presence of Aβ in the brain of these
mice, we tested the effect of an acute bolus of rt-PA in wildtype mice. Similarly, the single systemic
administration of rt-PA did not modulate the basal enzymatic activities of both plasmin (Fig. 3.3b)
and MMP2/9 (Fig. 3.3d) in the brain and the microvasculature of wildtype mice. It has been
reported that t-PA is also involved in neuronal remodeling and synaptic plasticity and mediates
Figure. 3.4. Chronic Activase® rt-PA administration modulates monocyte subpopulations frequencies
in the blood of APPswe/PS1 mice. Flow cytometry analysis (a-c) was performed to examine total
monocyte population frequency and subset frequencies in the blood of APPswe/PS1 mice. Activase® rt-PA
does not change total monocyte frequency in leukocytes (CD45+ cells) in the blood 24 hours after last
injection (a). A gating strategy (b) was thereafter used to discriminate inflammatory monocyte (Ly6CHigh)
and patrolling monocyte (Ly6CLow) subset frequencies in the total population of monocytes. Activase® rt-
PA significantly decreases Ly6CHigh monocyte subset frequency (c), without modulating Ly6CLow subset
frequency (d) in the blood 24 hours after last injection. Data are means ± SEM (n = 8-10 animals per group).
* P < 0.05 compared with saline treated group.
65
neuronal adaptation to metabolic stress via its proteolytic activity, by enhancing the levels of
synaptophysin, a major synaptic vesicle protein (183). Therefore, we next tested whether the rt-PA
enhanced the synaptic plasticity by enhancing synaptophysin levels. We first confirm that indeed
the protein expression level of synaptophysin has significantly decreased in the brain of
APPswe/PS1 mice compared to wildtype mice (Supplementary Fig. 3.4b). However, the
Activase® rt-PA regimen used in this study did not seem to affect synaptophysin levels
(Supplementary Fig. 3.4a, b) in the brain of APPswe/PS1 treated mice. These results indicate that
the mechanism underlying rt-PA-induced cerebral Aβ reduction and cognitive enhancement are
Figure. 3.5. Acute Activase® rt-PA administration modulates monocyte subpopulation frequencies in
the blood of wildtype mice. Flow cytometry analysis (a-f) examining total monocyte population frequency
and subset frequencies in the blood of wildtype mice 3 (a,c,e) and 24 hours (b,d,f). Activase® rt-PA does
not change the total monocyte frequency in circulating leukocytes (CD45+ cells) 3 (a) and 24 hours (b) after
a single systemic injection in wildtype mice. This treatment also failed to affect Ly6CHigh monocyte subset
frequency in the blood 3 (c) and 24 hours (d) after a single systemic injection. However, it significantly
increases Ly6CLow subset frequency 3 hours (e), but not 24 hours after last injection (f). Data are means ±
SEM (n = 8-10 animals per group) * P < 0.05 compared with saline treated group.
66
probably independent of the enzymatic activity of rt-PA, more precisely rt-PA-induced plasmin/
MMP2/9 activation.
3.5.4. Activase® rt-PA modulates monocyte population phenotypes in a transient manner
We next investigated the anti-inflammatory effects of rt-PA by analysing its possible role in
modulating monocyte populations. Three hours following the systemic administration of rt-PA,
total monocyte frequency significantly increased (Supplementary Fig. 3.5) without altering the
distribution of monocyte subpopulation in APPswe/PS1 mice. However, rt-PA did not alter total
monocyte frequency in the blood of APPswe/PS1 mice 24 hours following last injection (Fig. 3.4a),
but it induced a shift in monocyte phenotypes by reducing the frequency of Ly6CHigh inflammatory
subset (Fig. 3.4b,c) and by slightly increasing that of Ly6CLow patrolling subset (Fig. 3.4b,d). In
order to clearly address the direct effect of rt-PA on the population of monocytes in an Aβ-free
context, which has been shown to influence monocyte response (23), we used wildtype mice that
were injected with rt-PA. The injection of rt-PA was without effects on the total monocyte and
Ly6CHigh subset frequencies at 3 hours (Fig. 3.5a,c) or 24 hours (Fig. 3.5b,d), respectively, in
wildtype animals, although it significantly increased Ly6CLow subset frequency at 3 hours (Fig.
3.5e), but not at 24 hours (Fig. 3.5f).
3.5.5. The effects of Activase® rt-PA on resident microglia
Chronic rt-PA administration increased the number of resident microglia surrounding Aβ plaques
(Fig. 3.6a,b) as well as the number of Aβ-immunoreative resident microglia surrounding Aβ
plaques (Fig. 3.6a,c), which translates Aβ internalization (i.e phagocytosis) in vivo by these cells.
Interestingly, this phenomenon was associated with a significant global decrease in activation of the
stress kinases SAPK/JNK phosphorylation (Fig. 3.6d), without affecting p38 MAPK
phosphorylation (Fig. 3.6e). The enhanced microglial coverage of Aβ plaques was not due to an
increased infiltration of circulating monocytes and their subsequent differentiation into mature
microglia, as we did not detect any CD45High (blood-derived leukocytes)/ Iba1 (differentiated
microglia) double positive staining (i.e. blood-derived macrophages) within the cells surrounding
Aβ plaques (Fig. 3.7a). In addition, we did not detect any changes in the frequency of
CD11bHigh/CD45High (blood-derived macrophages) in the brain of APPswe/PS1 mice 3 and 24 hours
following rt-PA administration (Fig. 3.7b). Finally, these results were confirmed when we did not
detect GFP-positive cells in the brain of chimeric APPswe/PS mice treated with rt-PA (Fig. 3.7c).
Moreover, chronic rt-PA administration did not alter NF-κB signaling pathway activity that is
involved in microglial pro-inflammatory activation, which was unraveled by the unchanged gene
67
transcript expression levels of IκBα (316) (Supplementary Fig. 3.6c). These data suggest that rt-PA
Figure. 3.6. Chronic Activase® rt-PA administration increases the number of resident microglia-
associated to Aβ plaques and reduces the activation of stress-induced pathways. Immunofluorescence
staining (a-d) and western blot (e,f) analyses examining microglia association to Aβ plaques, Aβ
internalization by microglia and the activation of stress-related kinases in the brain of APPswe/PS1 mice,
10 weeks after systemic Activase® rt-PA administration. Triple 6E10/Iba1/DAPI immunofluorescence
staining shows an increased number of microglia (Iba1; green / DAPI; blue) surrounding Aβ plaques
(6E10; red) (a,b) and an increased number of Aβ-immunoreactive resident microglia (microglia
internalizing Aβ) (a,c). Moreover, Activase® rt-PA treatment decreases the phosphorylation levels of
SAPK/JNK (e) without affecting the phosphorylation levels of p38 MAPK (f). Optical densities were
corrected with β-actin levels. Data are means ± SEM (n = 4-6 animals per group, 4 sections representing
the rostral, middle and caudal levels of the hippocampus and overlaying cortex per animal’s). * P < 0.05,
** P < 0.01 compared with saline-treated group. Laser scan confocal images were acquired with a 60X
objective. Scale bar = 10 µm.
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might modulate resident microglia activity by enhancing their mobilization towards Aβ plaques, by
increasing their capacity to internalize Aβ, and by decreasing the stress associated to inflammation
without altering their phagocytic capacities.
Figure. 3.7. Activase® rt-PA administration does not influence blood-derived monocyte infiltration
into the brain parenchyma of APPswe/PS1 mice. Immunofluorescence staining (a,c) and flow cytometry
analysis (b) examining blood-derived monocyte infiltration into the brain parenchyma of APPswe/PS1.
Double Iba1/CD45 immunofluorescence staining shows the absence of CD45High/Iba1 positive cells (blood-
derived macrophages) (a) surrounding Aβ plaques in the brain of APPswe/PS1 mice, 10 weeks after
systemic Activase® rt-PA administration. Flow cytometry analysis shows that the frequencies of
CD11bHigh/CD45High cells (blood-derived macrophages) remain unchanged in the brain of APPswe/PS1
mice, 3 and 24 hours following a single Activase® rt-PA administration (b). Finally, Activase® rt-PA does
not trigger the infiltration of blood-borne GFP-positive cells into the brain of APPswe/PS1 chimeric mice
24 hours after a single Activase® rt-PA administration (c). In contrast, irradiation is triggers the infiltration
and differentiation (ramification) of blood-borne GFP-positive cells in the brain of APPswe/PS1 mice
(positive control) (c). Data are means ± SEM (n = 7-8 animals per group, 4 sections representing the rostral,
middle and caudal levels of the hippocampus and overlaying cortex per animal’s). Scale bar = 50 µm (a),
250 µm (c).
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3.5.6. Activase® rt-PA enhances BV2 microglial cell mobility and acts as chemoattractant
molecule in a LRP1-dependent manner
In order to fully address and decipher the effect of Activase® rt-PA on microglia, we performed a
series of experiments using the immortalized murine microglial cell line (BV2). The strategy
consisted on comparing BV2 cell stimulation with rt-PA along with two other molecules LPS and
interleukin (IL)-4, known to trigger the activation of these cells towards a pro-inflammatory
phenotype (LPS) or anti-inflammatory one (IL-4). rt-PA (0,1 nM) and IL-4 (5 ng/ml) exposure
enhanced microglial cell mobility in a similar manner (Fig. 3.8a), whereas they failed to induce
change in MMP2/9 (Fig. 3.8b) and LRP1, a receptor for t-PA (Fig. 3.8c). However rt-PA-induced
microglial cell mobility was LRP1 dependent, because LRP1 inhibition by RAP essentially
abolished rt-PA-induced cell mobility (Fig. 3.8d). We then verified the possible role of rt-PA as a
chemoattractant molecule that triggers microglial cell mobility and migration using a two chambers
transwell experimental setting (Fig. 3.8e) and found that rt-PA acted as a chemoattractant molecule
that mobilized microglial cells to the lower chamber that contains the molecule (Fig. 3.8f). This role
was mediated by LRP1 expressed on microglial cells since LRP1 inhibition by recombinant RAP
prevented these effects (Fig. 3.8f). Taken together these results underlie a key chemoattractant role
of rt-PA on microglial cells via LRP1. Activase® rt-PA dampers intracellular stress in BV2
microglial cells
The effects of Activase® r-tPA on the regulation of SAPK/JNK and p38 MPAK signalling pathway
were investigated in BV2 cells. In contrast to LPS (2 µg/ml), IL-4 and rt-PA failed to induce
SAPK/JNK phosphorylation (Fig. 3.9a), but they slightly increased p38 MAPK phosphorylation
(Fig. 3.9b). These effects of rt-PA were in contrast with the effect of LPS that causes a robust p38
signaling induction.
3.5.7. The effects of Activase® rt-PA on the phagocytic capacity and oxidative stress cascade
in BV2 microglial cells
Nitrite production and release by activated microglia is involved in microglial-derived oxidative
stress (324). Interestingly, rt-PA and LPS potently enhanced microglial cell phagocytic capacity 1
hour after stimulation (Fig. 3.9c). However, in contrast to LPS stimulation, rt-PA stimulation did
not trigger nitrite production and release by activated microglial cells (Fig. 3.9d). These results
suggest that rt-PA was able to enhance and preserve microglial cell phagocytic capacity without
triggering microglial-derived oxidative stress.
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Figure. 3.8. Activase® rt-PA modulates BV2 microglial cell activation in vitro. Cell migration assay
(a,d), gelatinase activity assay (b), western blot (c) and chemotaxis assay (e,f) analyses examining the
behavior of microglial cells after stimulation with Activase® rt-PA (0,1 nM), LPS (2 µg/ml), IL-4 (5 ng/ml)
and RAP (200 nM). Cell migration assay shows an enhanced migration of microglial cells 24 hours after
stimulation with rt-PA or IL-4 compared to control or LPS exposure (a). Gelatin activity assay shows that
Activase® rt-PA does not induce MMP2/9 activation in any conditions (b). Western blot analysis confirms
the unchanged expression levels of LRP1 (c). Cell migration assay shows that rt-PA-induced mobility is
LRP1-dependent, as LRP1 inhibition with RAP, decreases cell migration (d). The two chamber chemotaxis
assay provided evidence that rt-PA induces microglial mobilization towards a gradient of rt-PA present in
the lower chamber (e). Microglia cellsare mobilized by Activase® rt-PA in a LPR1-dependent manner 3
hours after stimulation (f). Optical densities were corrected with β-actin levels. Dark spheres represent BV2
cells. Data are means ± SEM (n = 3-4 independent experiments). * P < 0.05, ** P < 0.01, **** P < 0.0001
compared with control group. The descending arrow illustrates cell mobilization from the upper chamber
toward the lower chamber. Scale bar = 250 µm.
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3.5.8. The effects of Activase® rt-PA on the mobility and the phagocytic capacity of BV2
microglial cells is independent of its enzymatic activity.
In order to verify whether the observed effects of rt-PA are not associated to the its enzymatic
activity, we stimulated BV2 microglial cells with a full length mutated form of rt-PA that is
deprived of any enzymatic activity, rt-PA (S478A), in which alanine has been substituted for the
active-site serine. Interestingly, rt-PA (S478A) still potently enhanced microglial cell mobility (Fig.
3.9e) and phagocytic capacity (Fig. 3.9f). These results confirm once again the important role of rt-
PA as a cytokine in modulating microglial cell activity independently of its enzymatic activity.
3.6. Discussion
This study unravels a novel role for Activase® r-tPA in counteracting the progression of AD-like
pathology in APPswe/PS1 mice. These effects include Aβ clearance together with a slight increase
in the frequency of anti-inflammatory patrolling monocytes and a preserved phagocytic capacity of
resident microglia. It is noteworthy to mention that the Activase® r-tPA regimen used in this study
did not induce BBB breakdown, which was evaluated by albumin and IgG extravasation and the
expression levels of the tight junction protein Occludin. Moreover, Activase® r-tPA regimen did
not alter BBB function, which was evaluated by the expression levels of several transporters and
receptors involved in Aβ transport across the BBB, such as ABCB1 (50,55), LRP1 (54) and RAGE
(40). This study also shows that the systemic chronic administration of Activase® r-tPA decreased
Aβ plaque load and size, decreased the levels of soluble Aβ1-42 but not Aβ1-40 in the brain of
APPswe/PS1, and ameliorated the cognitive function of these mice. It is noteworthy to mention here
that memory retrieval in the T-water maze of rt-PA-treated APPswe/PS1 mice was significantly
improved indicating that rt-PA exerted beneficial effects on mice cognitive flexibility, which has
been shown to be impaired in AD (325,326).
The t-PA/plasmin system has been proposed to be involved in soluble and Aβ microaggregate
degradation either directly through the enzymatic activity of plasmin (162) or indirectly through the
enzymatic activity of t-PA-induced MMP2/9 activation (213–215). In parallel, several studies
outlined the role of the enzymatic activity of t-PA in promoting neuronal remodeling and synaptic
plasticity, consequently enhancing neuronal adaptation to metabolic stress, mainly by enhancing the
production of the synaptic vesicle protein synaptophysin (183). The Activase® r-tPA regimen used
in this study did not induce the activation of plasmin and MMP2/9, suggesting a rt-PA enzymatic-
independent mechanism involved in the clearance of soluble and insoluble Aβ. In line with other
reports(327), synaptophysin levels were significantly reduced in the brain of APPswe/PS1 mice
compared to wildtype littermates. However, rt-PA-enhanced cognitive function was not
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accompanied by, at least, detectable enhanced expression of synaptophysin in the brain of
APPswe/PS1 mice. This might be due to several factors, such as the differential expression of
synaptophysin in an age and brain region-dependent manner (328). Besides, several studies have
shown that some treatments enhanced the cognitive function of rodents, without modifying
synaptophysin levels (329). Finally, a recent study showed that Aβ disrupts the interaction between
synaptophysin and VAMP2 affecting the synaptic transmission (330). Our study indicates that the
enhanced cognitive functions of treated mice is, at least, independent of the enzymatic activity of t-
Figure. 3.9. Activase® rt-PA decreases BV2 microglial cell intracellular stress and preserves their
phagocytic capacity. Western blot (a, b), phagocytosis assay (c, f), ,Griess assay (d), cell migration assay
(e) analyses examining the microglia intracellular stress responses microglial mobilization and phagocytic
capacity after stimulation with Activase® r-tPA (0,1 nM), rt-PA (S478A) (0,1 nM), LPS (1 µg/ml) and IL-4
(5 ng/ml). Western blot analysis shows that rt-PA and IL-4 do not increase the phosphorylation of
SAPK/JNK, which was strongly increased in presence of LPS (a). The endotoxin also caused higher
phosphorylation levels of p38 MAPK than rt-PA and IL-4 (b) while the phagocytic capacity of microglial
cells remained similar between rt-PA and LPS (c). This response to LPS was associated with nitrite
production and release by microglial cells, which was absent in the presence of rt-PA (d). Cell migration
assay shows an enhanced migration of microglial cells 24 hours after stimulation with the mutated form of
rt-PA that is deprived of any enzymatic activity rt-PA (S478A) (e), Finally, the phagocytosis assay shows
that rt-PA (S478A) still enhances phagocytic capacity of microglial cells (f). Data are means ± SEM (n = 3
independent experiments). **** P < 0.0001 compared with control group.
73
PA in promoting neuronal remodeling and adaptation to metabolic stress, but rather probably linked
to the decreased levels of soluble and insoluble Aβ.
The relevance of t-PA system in AD has been outlined in some studies, suggesting it as a potential
therapeutic target (162,232). In the brain, t-PA is produced by brain endothelial cells and
throughout the brain parenchyma, namely by microglia (173). However, all experimental studies
have investigated endogenous t-PA with a focus of its well characterized proteolytic function. In
our study, Activase® r-tPA administration did not alter BBB function, thus suggesting that the rate
of Aβ transport across the BBB by specialized endothelial transporters were not affected. Therefore,
it is conceivable to propose that cerebral Aβ reduction following systemic rt-PA administration is
due to a dynamic and synergistic interaction between blood circulation and the brain. Importantly, t-
PA has been shown to act as an anti-inflammatory cytokine, independently from its proteolytic
activity (197). Therefore, Activase® r-tPA anti-inflammatory characteristics on circulating
monocytes were firstly investigated. In rodents, monocyte population is divided into two subsets,
the pro-inflammatory subset (i.e. Ly6CHigh) and the patrolling subset (i.e. Ly6CLow) (331). Both
monocyte subsets have been demonstrated to be involved in Aβ processing and play key roles in
AD pathogenesis and treatment strategies (23). Consistent with its anti-inflammatory
characteristics, rt-PA transiently decreased the frequency of pro-inflammatory monocyte subset and
induced a slight increase in the frequency of anti-inflammatory monocyte subset in blood
circulation. Although, rt-PA administration had little effects on key endothelial transporters and
receptors involved in specialized Aβ transport and elimination across the BBB, this does not
eliminate the fact that circulating monocytes are always capable to adhere to brain vasculature and
contribute in Aβ clearance. Indeed, very recently, our group demonstrated, by using a novel two-
photon intravital imaging approach to investigate the role of monocytes in the brain of live
APPswe/PS1 mice, that the anti-inflammatory monocyte subset adhered in a specific manner to Aβ
microaggregate-rich brain vasculature and efficaciously eliminated these microaggregates by
internalizing and transporting them from brain microvasculature to blood circulation (284). The
specific depletion of the anti-inflammatory monocyte subset in APPswe/PS1 mice increased overall
cerebral Aβ levels and worsened the cognitive function of these mice (284). Taken together, these
results demonstrate that rt-PA has to potential to specifically increase, although modestly, the
frequency of the anti-inflammatory monocyte subset that is involved in Aβ elimination from brain
vasculature, thus partly contributing in rt-PA-induced Aβ clearance.
Some reports showed that neuronal and microglial-derived endogenous t-PA triggers microglial cell
activation with diverse inflammatory responses (194,195,197,332). Importantly, intravenously
74
administered rt-PA has been shown to reach the brain parenchyma without altering the intact BBB
(322). However, little is known about the cytokine actions of exogenously administered rt-PA,
which imitates vascular-derived rt-PA production, on brain resident microglial cells in
neurodegenerative disorders, namely AD. Therefore, the effects of Activase® r-tPA on brain
resident microglia were also investigated. Activase® r-tPA chronic administration significantly
increased the number of resident microglia covering Aβ plaques, which translates an enhanced
mobility, invasion and mobilization of these cells towards Aβ aggregates. In parallel, Activase® r-
tPA administration significantly decreased the phosphorylation levels of SAPK/JNK in the brain of
treated mice. SAPK/JNK is potently activated by a variety of environmental stresses, including Aβ
(333). As such, these results outline a reduction in the environmental stresses in the brain of
APPswe/PS1 mice following rt-PA treatment. In parallel, NF-κB signaling pathway has been shown
to mediate the pro-inflammatory actions of microglia (334). Importantly, Activase® r-tPA did not
modulate IκBα gene transcript expression, which is an adaptor protein involved in controlling NF-
κB signaling pathway widely used as an indicator of NF-κB activity (316). These results indicate
that Activase® r-tPA administration mediated essentially a less pro-inflammatory phenotype of
resident microglia, which is consistent with t-PA anti-inflammatory characteristics (197).
The molecular mechanisms involved in Activase® r-tPA effects on microglial cells were next
investigated in vitro by using the BV2 microglial cell line. The BV2 cell line was chosen as it has
been reported to be a valid substitute for primary microglial cell culture in several experimental
settings (335). Activase® r-tPA stimulation enhanced microglial cell mobility and invasion in a
similar manner as IL-4 but in contrast to LPS. This effect was dependent on the interaction between
rt-PA and its receptor on microglial cells, LRP1, as the inhibition of the latter with recombinant
RAP essentially abolished the effects of rt-PA. Moreover, this invasion did not depend on the
enzymatic activity of MMP2/9. The diffuse nature of endogenous t-PA and exogenously
administered rt-PA within the brain parenchyma prompted us to test its chemoattractant properties
(332). Indeed, by using the two chambers transwell experiment, Activase® r-tPA triggered
microglia cell mobilization into the chamber containing the molecule, which was LRP1-dependent,
as microglial cell incubation in the upper chamber with RAP totally abolished the chemoattractant
characteristics of rt-PA. In parallel, microglial cell stimulation with rt-PA and IL-4 did not induce
SAPK/JNK phosphorylation, a kinase involved in mediating the pro-inflammatory actions of
microglia (336), whereas LPS potently induced it. Moreover, rt-PA and IL-4 slightly increased p38
MAPK phosphorylation, another kinase that is also associated to microglial stress (337), whereas
LPS induced a robust increase in p38 MAPK phosphorylation. Finally, Activase® r-tPA stimulation
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enhanced and preserved the phagocytic capacity of microglial cells, without mediating nitrite
production that is involved in microglial-derived oxidative stress (324). These results clearly
demonstrate that rt-PA enhanced the phagocytic capacity of microglial cells without generating
nitrite production. It has been demonstrated that a pronounced production of nitrites by activated
microglial cells exacerbates the pro-inflammatory microenvironment that occurs in AD, thus
contributing to the decreased efficiency of resident microglia to clear Aβ (312,338). Taken together
these results suggest that Activase® r-tPA reduces microglia pro-inflammatory actions and
enhances their phagocytic capacity in the brain of APPswe/PS1 mice. Some in vivo experimental
studies have investigated the implication of the t-PA/plasmin system in AD pathogenesis and
treatment. However, all these studies focused essentially on the implication of the enzymatic
activity of t-PA. For example, it has been shown that the depletion of endogenous t-PA accelerates
and aggravates the pathogenesis of AD in Tg2576 mice (319). In addition, when synthetic Aβ was
injected into the brain of transgenic mice lacking t-PA or plasminogen, Aβ deposits persisted longer
and resulted in an exacerbated neuronal damage compared to wildtype littermates (162). In line
with thoses previous reports, our study provides new insights regarding the potential of this system
in AD treatment, by outlining a new proteolytic-independent mechanism of exogenously
administered rt-PA. On the other hand, some ex vivo and in vitro data reported that endogenous t-
PA mediates Aβ-induced neurotoxicity (224), and triggers the pro-inflammatory activation of
microglial cells in a glial cell culture (339) in the context of AD. Taken together, these
contradictory reports outline a very complex interaction between the t-PA/plasmin system and Aβ,
which seem to be dependent on the context under which this system is investigated in AD. As such,
we believe that more studies should be performed in order to better elucidate the contribution of this
system in AD and its potential as a therapeutic target.
3.7. Conclusion
It has been shown that a defective production of monocytes was accompanied by an increased
accumulation of soluble Aβ in the brain of APPswe/PS1 mice, which was closely associated to the
onset of cognitive decline (23). Macrophage-colony stimulating factor (m-CSF) treatment prevented
and restored the cognitive decline of mice by increasing monocyte production in blood circulation
(23). In parallel, microglia have been shown to be recruited to Aβ plaques, and to internalize and
degrade Aβ microaggegates (313). However, over time, the capacity of resident microglia to clear
Aβ decreases and becomes inefficacious due to the presence of an exacerbated pro-inflammatory
microenvironment (68). Nonetheless, the immunostimulation of resident microglial cells by
exogenous agents, such as the detoxified ligand monophosphoryl lipid A (MPL), reinforced Aβ
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clearance by microglial cells and improved the cognitive functions of APPswe/PS1 mice (340).
These observations suggest that the therapeutic strategies that aim at boosting monocyte production
and microglial cell activity with moderate inflammatory responses are very promising, and
constitute attractive avenues in combating AD progression. Therefore, our study suggests that the
FDA approved drug Activase® rt-PA might constitute a new potential treatment for AD with all
these very interesting features.
3.8. Acknowledgments
We specially thank Mr. Mohammed Filali for his technical expertise in beahavior test. We thank,
Mrs. Nataly Laflamme, Mrs. Marie-Michèle Plante and Mr. Paul Préfontaine for their technical
support.
3.9. Funding
This work was supported by the Canadian Institutes in Health Research (CIHR) and Canadian
Stroke Network.
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Chapitre 4
4. Discussion
Lors du chapitre 2, nous avons reporté un déclin cognitif plus important chez les souris
hypoperfusées comparé aux souris de la même portée non-hypoperfusées. Nos résultats démontrent
que, suite à la SCCH, l’apport en glucose diminue ce qui induit un dysfonctionnement microglial,
soit une réduction du recrutement et de l’activité des microglies autour des plaques. L’altération de
l’activité microgliale compromet la dégradation de l’Aβ et donc, contribue à l’augmentation du
nombre de plaques amyloïdes. L’hypoperfusion entraîne également une diminution de l’activation
de la voie ERK, impliquée dans la survie (297,306,307) et la croissance cellulaire (300,306). Ces
phénomènes contribuent indirectement et directement au déclin cognitif (Fig. 4.1).
Après une période de 15 semaines, les souris soumises à la SCCH démontraient une dysfonction de
leur mémoire spatiale et non spatiale sans présenter de déficits moteurs. En ce sens, l’altération de
la mémoire induite par l’hypoperfusion est un phénomène qui a été décrit à maintes reprises chez
des modèles animales non-mutants (147,149,151,153,154,156,341). Utilisant le modèle du 1VO
(137,138) et du BCAS (139,144), plusieurs ont décrit une aggravation du déclin cognitif des souris
transgéniques sur-exprimant l’APP humaine. En accord avec ces récents travaux menés chez les
souris Alzheimer (137–139,144), nos résultats confirment la participation de l’hypoperfusion dans
Figure 4.1. L’effet de l’hypoperfusion cérébrale chronique sévère (SCCH) sur le cerveau des
APPswe/PS1. Suivant l’hypoperfusion, le CBF et le glucose diminuent (1). L’environnement faible en
glucose créé induit une dysfonction microgliale (2) laquelle entraîne une augmentation du nombre de
plaques amyloïdes (3). La SCCH induit également une réduction de l’activité de la voie ERK1/2 qui est
associée avec la survie cellulaire et la croissance des neurites (4). La hausse de la déposition de l’amyloïde
pourrait également contribuer à l’altération de ERK (4). L’augmentation du nombre de plaques séniles et
l’altération de ERK contribuent au déclin de la mémoire spatiale et non spatiale. Afin de simplifier la
figure, l’amyloïde n’est pas représentée.
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la progression de la AD (42). De plus, notre étude indique que les souris transgéniques soumises à
la SCCH ne présentent pas de mort neuronale ou de rupture de la BBB. Ainsi, le déclin cognitif des
souris hypoperfusées n’est pas induit par des dommages de la matière blanche ce qui indique qu’il
ne s’agit pas d’une ischémie (139,149,151–157). De ce fait, nos résultats suggèrent que la SCCH
promeut une oligémie qui, tel que décrit par des travaux antérieurs, entraîne un dysfonctionnement
neuronal (138,140). Par conséquent, ce nouveau modèle, jumelant une BCAO transitoire de courte
durée et une 1VO permanente, modéliserait une oligémie qui serait plus sévère que le modèle 1VO.
Toutefois, cette théorie devrait être confirmée par la caractérisation des changements du CBF, soit
par l’imagerie par résonance magnétique ou par vélocimérie laser. En effet, ces deux tehniques
d’imagerie, qui ne nous était pas disponible pour cause de coûts et/ou de disponibilité,
permetteraient de quantifier et monitorer le changement du CBF suivant la SCCH. De plus,
l’imagerie par résonance magnétique permettrait également d’identifier les régions plus atteintes par
le changement du CBF. Ces régions cérébrales s’avèrent alors intéressante d’étudier plus
amplement par immunofluorescence ou immunohistochimie et ce, selon leur hémisphère.
De plus, le type d’hypoperfusion module l’accumulation et la déposition de l’Aβ. Selon notre
modèle de SCCH, le déclin cognitif était également accompagné par une augmentation significative
du nombre de plaques séniles à l’hippocampe sans changer la concentration d’amyloïde soluble
cérébrale. Cette corrélation a précédemment été établie par Pimentel et al suivant l’oligémie légère,
la 1VO, chez les APPswe/PS1 (138). Une autre étude a également décrit cette corrélation suite à
une sténose provoquant un stress ischémique chez les Tg swe/dutch/iowa (144). En contraste, Koike
et al ont effectué une BCAO transitoire de 4 minutes sur des souris transgéniques 3xTg-AD. Après
48 heures, ils ont observé l’augmentation de l’expression de BACE-1 qui induit la production de
l’Aβ et l’accumulation significative de l’Aβ soluble (140). L’induction de l’expression de la β-
sécrétase se produit habituellement suite à l’hypoxie qui accompagne généralement l’ischémie (89).
Cependant, ces changements n’ont pas été accompagnés de dommages de la matière blanche
suggérant une oligémie hypoxique (140). Au contraire, notre modèle n’induit pas de changement de
la concentration de l’amyloïde soluble, mais une élévation du nombre de plaques. Bien que
globalement notre étude et la leur démontrent l’altération de l’amyloïde, ces deux phénomènes
impliqueraient des mécanismes différents. Étant donné l’absence de changement de la quantité
d’amyloïde soluble, la SCCH ne semble pas moduler directement l’expression de l’Aβ suggérant
que l’accumulation de l’amyloïde s’effectue, soit par son entrée de la périphérie (55) ou l’altération
de son élimination (64). En effet, lors de travaux récents menés par notre laboratoire, ElAli et al ont
démontré que l’oligémie, provoquée par la 1VO chez des souris sauvages, promeut l’entrée de
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l’amyloïde périphérique (injectée par voie intrapéritonéale) au cerveau contribuant à sa déposition
(55). Par conséquent, dans le modèle de la SCCH sur les APPswe/PS1, l’augmentation de la
déposition de l’Aβ pourrait provenir de la périphérie. Cependant, puisqu’il ne s’agissait pas de l’un
des objectifs initiaux de ce projet, nous n’avons pas visualisé le transport de l’amyloïde par
microscopie intravitale (55). Ainsi, nous ne pouvons pas confirmer l’entrée de l’Aβ au cerveau.
Nous aurions également pu confirmer l’implication de l’augmentation du nombre d’agrégats
d’amyloïde sur les changements mnésiques observés en comparant par des tests de comportement
des souris non-mutantes hypoperfusées et APPswe/PS1 hypoperfusées. Considérant les
changements d’amyloïde suivant la SCCH, la comparaison entre ces deux groupes permettraient de
vérifier si l’altération de la mémoire et de l’apprentissage est accentué chez les APPswe/PS1 et si
celle-ci implique un mécanisme dépendant de l’amyloïde. Toutefois, pour des raisons éthiques,
nous avions exclus le groupe contrôle non-mutant afin de réduire au maximum le nombre
d’animaux inclus dans le protocole, puisque l’hypoperfusion est reconnue comme causant des
problèmes de mémoire (147,149,151,153,154,156,341).
L’accumulation de l’amyloïde peut également provenir de l’altération de l’une des voies
d’élimination. Par conséquent, nous nous sommes intéressés particulièrement à l’effet de la SCCH
sur l’une des voies de dégradation de l’Aβ, soit la phagocytose par les cellules immunitaires. Nous
avons donc déterminé la fréquence des populations de monocytes par cytométrie en flux. Comparé
aux souris non-hypoperfusées, les souris hypoperfusées semblent posséder davantage de monocytes
patrouilleurs, sans changement de la fréquence des monocytes totaux et inflammatoires. Plusieurs
travaux ont constaté que l’hypoperfusion déclenche un stress vasculaire qui induit le recrutement
des monocytes contribuant au remodelage vasculaire (292) et à la survie neuronale (293). Durant la
phase aiguë de l’ischémie, les monocytes inflammatoires sont recrutés afin d’éliminer les débris
produits (283,294,298). Plus tard, lors de la phase chronique de l’hypoperfusion, les monocytes
patrouilleurs (Ly6CLow) sont, à leur tour, recrutés (299). Nahrendorf et al décrivirent l’implication
de ces derniers dans les processus réparateurs lors de l’infarctus du myocarde (294). En ce sens, la
tendance de l’augmentation de la fréquence des Ly6CLow suivant la SCCH pourrait être promue par
des évènements moléculaires et cellulaires induits par le stress vasculaire. De manière intéressante,
des résultats non-publiés de cette étude (Fig. supplémentaire 4.1) ont démontré une réduction
significative de l’amyloïde vasculaire suggérant l’élimination de l’Aβ par les Ly6CLow. Cette
élimination serait limitée à leur interaction de l’Aβ, puisqu’aucune infiltration des monocytes n’a
été reportée. En ce sens, Michaud et al ont démontré que la 1VO augmente l’adhésion et le
rampement des Ly6CLow ce qui contribue à l’élimination de l’amyloïde vasculaire chez les
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APPswe/PS1 (284). Ils ont également prouvé que la déplétion du facteur de différenciation des
Ly6CLow (Nur77–/–), Nr4a1, augmente l’aire et le nombre des plaques amyloïdes (284). De plus, des
travaux chez les APPswe/PS1/CCR2–/–, déficiente en monocytes inflammatoire, ont reporté
l’importance des Ly6CHigh dans l’élimination de l’Aβ en observant une progression accélérée de la
pathologie (285,290). Toutefois, nos résultats suggèrent aucune implication des monocytes
inflammatoires. Bien que cette tendance ne soit pas significative, il s’agit d’une tendance forte,
considérant que l’échantillonnage était limité et que la fréquence des monocytes varie d’un individu
à l’autre. Le suivi longitudinal de la variation des populations des monocytes aurait pu permettre
d’évaluer plus exactement ces changements. Nous supposons que l’augmentation des monocytes
patrouilleurs à la suite de la SCCH contribue à la maintenance de la BBB en réponse au stress
vasculaire, en plus de contribuer à l’élimination de l’amyloïde vasculaire. Toutefois, d’autres
investigations complémentaires sont nécessaires afin de confirmer l’implication des monocytes
patrouilleurs dans l’élimination de l’amyloïde vasculaire. L’étude de souris APPswe/PS1 «knock-
out» Nur77–/– ou chimériques (Nur77–/–→APP/PS1) hypoperfusées permettraient d’évaluer l’impact
de la déplétion des Ly6CLow sur l’élimination de l’amyloïde vasculaire (342).
Outre les monocytes, les microglies contribuent également l’élimination de l’Aβ cérébrale. Nous
avons observé, chez les souris soumises à la SCCH, une réduction significative du nombre de
microglies recrutées aux agrégats et du nombre activées (CD68+) suggérant un dysfonctionnement
microglial. En ce sens, Hefendeh et al ont décrit des changements morphologiques chez les
microglies âgées, attribués un état dysfonctionnel (343). Au cours du vieillissement normal, le CBF
se réduit (95) ce qui limite l’apport en glucose et en oxygène (129,131). Dans ce contexte, la
réduction du glucose peut induire un dysfonctionnement métabolique des cellules exigeantes
énergétiquement (95,130) dont les microglies font partie (279). Limitant la production d’ATP (132),
la déficience en glucose pourrait compromettre la phagocytose (304). Le cas échéant, les microglies
entreraient dans un état dysfonctionnel, puisque celles-ci sont incapables de subvenir à leur besoins
énergétiques. Afin de confirmer ce mécanisme, l’effet de la déficience en glucose a été étudié in
vitro chez les BV2, une lignée cellulaire murine. Tel que présumé, nos résultats ont démontré qu’un
microenvironnement appauvri en glucose altère l’activité globale, l’activation et la capacité
phagocytique des microglies. Cet hypométabolisme microglial confirme le dysfonctionnement des
microglies observé in vivo, en plus de favoriser la déposition de l’amyloïde par la perte de capacité
phagocytique. Ainsi, la SCCH dévoile un nouvel effet de l’hypoperfusion sur la fonction des
microglies. En effet, les travaux antérieurs ont reporté l’altération du comportement microglial 3
mois suivant l’ischémie. Toutefois, ils y ont observé une activitée accrue des microglies dans la
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région CA1 de l’hippocampe corrélant avec la phagocytose de neurones apoptotiques (151,156). Au
contraire, l’oligémie, causée par la SCCH, induit ni la mort neuronale, ni l’activation microgliale,
mais promeut un dysfonctionnement de ces cellules immunitaires. Considérant l’augmentation du
nombre de plaques, nos résultats démontrent, pour la première fois, l’hypoactivité des microglies ce
qui contribue à l’accumulation et à la déposition de l’amyloïde.
D’autre part, la SCCH induit la réduction de la phosphorylation de ERK globale au cerveau pouvant
indiquer un dysfonctionnement neuronal. En effet, la voie de ERK module entre-autres les effets
neuroprotecteurs de plusieurs facteurs neurotrophiques (297,300,307). Son altération est reconnue
pour induire une altération neuronale, soit de la survie (297,300–302,306,307), de la prolifération
(344) et de la croissance des neurites (306). Chez des souris sauvages hypoperfusées, l’altération
des protéines impliquées dans la fonction neuronale a été reporté à maintes reprises pour les
neurotrophines (145,147) la voie PI3K/Akt (147,153), la synaptophysine, GAP-43 et MAP-2 (146).
La réduction de l’activité de ERK pourrait également influencer l’inflammation (295,296) et
augmenter la production des dérivés réactifs de l’oxygène (303). Toutefois, l’évaluation de
l’expression de ERK étant faite sur des homogénats de cerveau, il est davantage probable que la
réduction d’activation de ERK contribue au dysfonctionnement neuronal. Dans le contexte de la
AD, cela aggrave le déclin cognitif.
Par la suite, dans le chapitre 3, nous avons décrit un nouvel effet de l’Activase® rt-PA sur les souris
APPswe/PS1. Lorsqu’administré hebdomadairement avant le développement des premiers
symptômes de la AD, le rt-PA retarde la progression de la maladie. En effet, sans altèrer l’intégrité
ou la fonction de la BBB, le rt-PA améliore la fonction cognitive et réduit l’accumulation de
l’amyloïde. Par son action de cytokine, le rt-PA module le phénotype des monocytes et des
microglies résidentes vers un phénotype anti-inflammatoire. In vivo, les microglies sont davantage
recrutées au niveau des plaques, ce qui indique un gain de mobilité induit par rt-PA confirmé par
l’étude in vitro. Outre l’augmentation de leur mobilité, l’étude in vitro de rt-PA sur les microglies,
les BV2, a permis de démontrer que le rt-PA se lie à LRP1 et induit l’activation de la voie de
signalisation MAPK p38 ce qui favorise la phagocytose sans induire de stress oxydatif (nitrite,
SAPK/JNK). Cet effet est également préservé lors de la stimulation des BV2 avec le rt-PA muté au
niveau de son site enzymatique (rt-PA S478A) confirmant l’absence de l’implication de la fonction
enzymatique du t-PA (Fig. 4.2).
Avec l’âge et la AD, l’expression du t-PA diminue drastiquement (162,226,231,233,234). À
maintes reprises, des travaux ont suggéré l’importance d’explorer le système de t-PA en tant que
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cible thérapeutique potentielle (162,229,231,232). Dans cette étude, nous nous sommes intéressés à
l’effet du rt-PA sur le cerveau des souris transgéniques APPswe/PS1. Pour ce faire, nous avons
administré 5mg/kg de l’Activase® rt-PA à chaque semaine pendant 10 semaines à des souris
APPswe/PS1 âgées de 4 mois. Comparées aux souris traitées avec du salin, l’administration
chronique de rt-PA chez les APPswe/PS1 induit une amélioration des facultés cognitives au test du
Water-T maze. Le rt-PA entraîne également la réduction de la taille et du nombre de plaques
amyloïdes du cortex et de l’hippocampe, en plus de réduire la concentration d’Aβ1-42 soluble. En ce
sens, des études antérieures ont également établi que l’activation de t-PA par l’invalidation génique
des inhibiteurs endogènes de t-PA réduit l’accumulation de l’Aβ cérébrale des souris transgéniques
Alzheimer (229,230). De plus, Oh et al ont récemment démontré que la déplétion de t-PA chez des
souris Tg2576 aggrave le déclin cognitif et promeut l’accumulation de l’Aβ (232). Suite à
l’administration du rt-PA, aucune rupture de la BBB n’a été observée. Le rt-PA a également eu
aucun effet sur l’expression de transporteurs et récepteurs impliqués dans le transport de l’Aβ, soit
ABCB1 (50,55), LRP1 (54) et RAGE (40). De ce fait, le délai de la progression de la AD n’est pas
induit par un changement de la BBB, mais par un mécanisme impliquant la fonction enzymatique
ou cytokine de t-PA.
Dans la présente étude, l’administration de rt-PA (5mg/kg) n’induit pas l’activation de la plasmine
ou des MMP2/9 suggérant l’élimination de l’Aβ par un mécanisme indépendant de son activité
Figure 4.2. L’effet de l’administration hebdomadaire de l’Activase® rt-PA sur le cerveau des
APPswe/PS1. Suite à l’administration du rt-PA chronique, aucun changement de la BBB n’est observé
(1). Le rt-PA induit la diminution de la fréquence des monocytes inflammatoires (1), en plus d’un gain
de la mobilité et de la phagocytose des microglies (2). L’augmentation de la fonction microgliale
contribue à réduire l’aire et le nombre de plaques amyloïdes, de même que la quantité d’Aβ1-42 soluble
(3). Le rt-PA active les microglies qui élimine efficacement l’Aβ et ce, sans promouvoir
d’augmentation de la production des dérivés réactifs à l’oxygène (2). Ainsi, le rt-PA retarde la
progression de la AD. Afin de simplifier la figure, l’amyloïde n’est pas représentée.
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enzymatique de t-PA. Exprimé et activé à proximité des plaques d’amyloïde (162,223), le t-PA est
impliqué dans la dégradation de l’Aβ (162). En effet, le t-PA la stimule directement par la plasmine
(225) et indirectement par les MMP2/9 (213–215) la dégradation de l’amyloïde. Or, nous n’avons
pas observé leur activation à la suite de l’administration de la dose de rt-PA. De plus, l’action
protéase de t-PA ne contribue pas à l’amélioration des fonctions cognitives, puisqu’aucun
changement de l’expression de la synaptophysine n’est observé. Tout comme les APPswe/PS1 non-
traitées, celles traitées avec t-PA présentent une diminution de l’expression de la synaptophysine.
En contraste, plusieurs travaux ont mis en évidence la contribution de t-PA à la plasticité synaptique
et à la LTP (183,198,199). En effet, la fonction protéolytique de t-PA promeut la sécrétion de
vésicules contenant la synaptophysine (183) de même que la conversion de proBDNF et proNGF en
leur forme active (198,199). Cependant, malgré l’amélioration des fonctions cognitives, plusieures
études ont démontré l’absence de changement des niveaux de synaptophysine suggérant qu’un autre
mécanisme soit impliqué (329). Ainsi, l’effet bénéfique de rt-PA sur la faculté cognitive serait
promu par la réduction de l’Aβ soluble et insoluble plutôt que par l’activité protéolytique. Cette
théorie devra être confirmer par l’emploi de souris témoins non-mutantes non-traitées et traitées
avec rt-PA (5 mg/kg). Ainsi, l’absence de changement de la capacité d’apprentissage et de
mémorisation indiquerait que l’amélioration cognitive observée chez les APPswe/PS1 est
principalement reliée à la diminution d’Aβ.
Indépendamment de son activité enzymatique, le t-PA peut également agir en tant que cytokine
parfois anti-inflammatoire (197) ou inflammatoire (191–194). Ainsi, nous avons étudié l’effet du rt-
PA sur les cellules immunocompétentes. Nous avons d’abord défini l’effet du rt-PA sur les
monocytes par cytométrie en flux. À la suite de l’administration chronique de l’Activase® rt-PA,
celui-ci induit un profil anti-inflammatoire des monocytes circulants induisant l’augmentation de la
fréquence des Ly6CLow. L’effet anti-inflammatoire du rt-PA est également observé suivant
l’administration d’une dose unique. En effet, la diminution des monocytes Ly6CHigh est observée 3
heures plus tard, mais ne perdure pas au-delà de 24 heures. L’analyse de marquage
d’immunofluorescence et de cytométrie sur le cerveau des souris APPswe/PS1 traitées et non-
traitées a permis de constater l’absence d’infiltration des monocytes au cerveau. Précédemment, il a
été reporté que l’action cytokine de rt-PA module le recrutement des macrophages (196). Malgré
l’absence de recrutement des monocytes au cerveau, les monocytes circulants peuvent contribuer à
l’élimination de l’amyloïde (284,285,290). Tel que reporté par Michaud et al, les monocytes
Ly6CLow adhèrent la BBB ce qui leur permet d’internaliser et de dégrader des micro-agrégats
d’amyloïdes (284). De plus, l’invalidation génique de cette population de monocytes chez des
84
souris transgéniques (APPswe/PS1/Nur77–/–) aggrave les troubles cognitifs et augmente
l’accumulation de l’Aβ (284). Par conséquent, l’Activase® rt-PA promeut un profil anti-
inflammatoire des monocytes circulants qui peuvent contribuer à l’élimination de l’amyloïde.
Nous avons également reporté cet effet anti-inflammatoire chez les microglies. Lors de
l’administration chronique de rt-PA, l’immunoréactivité et le recrutement des microglies aux
plaques amyloïdes augmentent significativement et ce, en absence d’inflammation. En effet,
l’administration de rt-PA ne module pas l’expression de l’ARNm d’IκBα, l’inhibiteur de NFκB
(316) impliqué dans la réponse inflammatoire (334). Qui plus est, rt-PA induit une réduction
significative de l’activation globale de la voie SAPK/JNK au cerveau, une kinase qui module la
réponse pro-inflammatoire (336). Grâce à l’étude in vitro avec les BV2, nous avons pu déterminer
que le rt-PA augmente la mobilité cellulaire ce qui correspond à l’augmentation du recrutement des
microglies aux plaques observée in vivo. Par la suite, nous avons mis en évidence l’absence
d’activation des MMP2/9 suivant l’exposition des BV2 au t-PA. Exprimant fortement LRP1 par les
microglies, le t-PA peut les moduler par ce récepteur (322). Nous avons donc déterminé si son
action sur la mobilité de t-PA est dépendente de LRP1 par la co-stimulation de t-PA et RAP, un
ligand de LRP1 (345). Ainsi, nous avons confirmé l’implication de LRP1 qui induit la mobilité
microgliale. De plus, rt-PA agit également comme molécule chimio-attractante sur les microglies
selon un mécanisme impliquant LRP1. Mise à part cet effet moléculaire, nos résultats décrivent
l’activation de la voie MAPK p38, une kinase associée au stress microglial (337), suivant
l’exposition à rt-PA. Cette activation a d’ailleurs été associée à un gain de la capacité phagocytique
des microglies, sans augmenter de la production des nitrites. Globalement, le comportement des
microglies stimulées par le rt-PA se rapproche de celui des microglies stimulées par l’IL-4,
représentant un profil anti-inflammatoire. Comme l’IL-4, le rt-PA n’induit pas la phosphorylation
de SAPK/JNK. À l’inverse, le LPS induit la phosphorylation robuste de SAPK/JNK et de MAPK
p38, en plus d’induire de la production de nitrites, soit l’indicateur du stress oxydatif chez les
microglies (324). De plus, le rt-PA S478A, muté à son site enzymatique, possède le même effet que
rt-PA non-muté sur la mobilité et la phagocytose des microglies, réaffirmant l’absence
d’implication de l’activité protéolytique du t-PA. Par conséquent, nos résultats démontrent que le rt-
PA induit un profil anti-inflammatoire des microglies ce qui augmente leur mobilité et leur capacité
phagocytique selon un mécanisme LRP1 dépendant. Ce gain de la capacité phagocytique permet
notamment aux microglies d’éliminer plus efficacement l’Aβ du cerveau et ce, sans induire la
production de nitrites qui aggrave le déclin cognitif lorsqu’elle est importante (312,338). En accord
avec les travaux de Stringer (197), nous décrivons l’action de cytokine anti-inflammatoire du rt-PA.
85
Cependant, plusieurs travaux ont décrit l’induction d’un profil pro-inflammatoire suivant
l’administration du t-PA (191–194). Dans le contexte de la AD, d’autres études ex vivo et in vivo
ont également démontré que le t-PA endogène module la toxicité de l’Aβ (224), en plus d’induire
l’activation pro-inflammatoire des microglies (339). Ainsi, ces résultats contradictoires suggèrent
un mécanisme d’action complexe de t-PA possiblement modulé par la dose administrée. Il serait
alors nécessaires d’étudier plus en profondeur l’effet de l’administration de l’Activase® rt-PA chez
les souris APPswe/PS1 dans une optique pharmacologique. En s’inspirant des travaux de Benchane
et al. (2005), nous pourrions étudier la pharmacocinétique de rt-PA administré, soit la
biodisponibilité de t-PA biotinylé au cerveau par immunohistochimie contre la biotine. Il serait
également pertinent de caractériser l’effet de la dose (Ex. 0 à 10 mg/kg) et la fréquence
d’administration (Ex. 1 à 7 injections/semaine) ce qui permettrait de déterminer les conditions
minimales recquise pour induire les effets bénéfiques que nous avons caractérisé et la dose
maximale administrable sans effets secondaires, soit définir la fenêtre thérapeutique du rt-PA chez
les souris APPswe/PS1. Dans une optique plus mécanistique, nous devrions confirmer et quantifier
l’interaction entre rt-PA marqué avec un fluorochrome ou biotinylé et son récepteur, LRP1, selon la
dose administrée par co-localisation des marqueurs en immunofluorescence ou par le transfert
d’énergie entre molécules fluorescente mesuré à l’aide de la cytométrie en flux. De plus, agissant
par l’entremise de LRP1, l’effet de l’Activase® rt-PA ne se limite toutefois pas aux monocytes et
aux microglies. L’évaluation de l’effet de l’administration de rt-PA sur les astrocytes et les
péricytes devront être effectuées afin de confirmer l’intérêt de rt-PA comme cible thérapeutique.
87
Chapitre 5
5. Conclusions et perspectives
Tout d’abord, nous avons démontré que le remodelage de la NVU, soit l’altération de l’activité
microgliale et de la voie ERK, provoqué par la SCCH aggrave le déclin cognitif des souris
transgéniques Alzheimer (Chapitre 2). Par la suite, nous avons observé les bénéfices accompagnés
de la compensation à faible dose du t-PA sur la NVU et sur la fonction cognitive des souris
APPswe/PS1. Par son action de cytokine, le t-PA active les microglies vers un profil anti-
inflammatoire (Chapitre 3). Considérés ensemble, nos résultats illustrent la dualité du remodelage
de la NVU ; pouvant prendre part à la cascade pathogénique de la AD, mais également servir de
cible thérapeutique pour retarder sa progression.
Cette dualité souligne l’importance de comprendre les processus cellulaires et moléculaires induits
dans la NVU lors de la AD. Cette compréhension s’avère nécessaire au développement de
traitement efficace considérant la complexité de la AD. L’étude parallèle des troubles vasculaires et
de la AD constitue une première approche efficace en cette direction. Le nouveau modèle, la SCCH,
que nous avons développé, est d’autant plus intéressant qu’il se rapproche davantage des troubles
vasculaires observés avec la MCI que l’ischémie (124). Ce modèle est également facile à illustrer in
vitro par l’exposition des cellules à un milieu faible en glucose. Bien qu’à présent, aucune altération
des astrocytes et des péricytes n’a été observée suivant l’oligémie (346), l’étude approfondie de
l’impact de la SCCH sur ces cellules devra être effectuée, puisqu’il s’agit d’une oligémie plus
sévère. Celle-ci permettrait de déterminer si un hypométabolisme des cellules de la NVU, autres
que les microglies, se développe. Étant observé avec l’âge (162,175,198,233,234), l’altération de
l’expression et/ou l’activité du t-PA pourrait se produire après la SCCH, puisqu’il est reconnu que
l’hypoperfusion cérébrale se développe également avec le vieillissement (130,142).
D’autre part, la combinaison de la SCCH et de l’administration du t-PA ou du glucose sur des
APPswe/PS1 constituent également des pistes à explorer pour le développement de traitement
transposable chez l’humain. En traitant les souris transgéniques hypoperfusées avec le t-PA ou du
glucose (55), nous nous attendons à amoindrir ou même renverser l’effet de l’hypoperfusion en,
respectivement, stimulant l’activation alternative des microglies et amoindrissant leur
hypométabolisme. De plus, il a été proposé que, lors de l’ischémie, rt-PA protège les neurones par
un mécanisme indépendant de la conversion de la plasmine (200,347). Une récente étude, menée
88
par An et al, a démontré l’effet protecteur de t-PA contre le stress métabolique et l’excitoxicité par
un mécanisme indépendant de son activité enzymatique (348). En effet, suivant le stress
métabolique, ils ont reporté la sécrétion de t-PA par les neurones et les astrocytes ce qui induit le
recrutement de GLUT1, le transporteur du glucose, et module la prise de glucose par les astrocytes
et les cellules endothéliales (348). Ainsi, ce nouvel effet de t-PA décrit par An et ses collaborateurs
pourrait permettre de restaurer la balance énergétique suivant la SCCH par le recrutement de
GLUT1.
Plusieurs traitements de la AD développés ciblent les cellules immuno-compétentes lesquelles
peuvent éliminer efficacement l’Aβ. Toutefois, l’activité immune peut agir comme une épée à
double tranchant ; apte à éliminer l’Aβ, mais nuisible lorsque sur-stimulé (349). Par conséquent,
certains scientifiques ont tenté de supprimer la réaction inflammatoire, ce qui ne contribue pas
nécessairement à l’arrêt de l’inflammation-même (350). Pour cette raison, notre équipe a suggéré de
s’intéresser à la mobilisation des monocytes comme voie thérapeutique (313,314,351), par exemple
par le traitement de M-CSF ou G-CSF. L’administration du M-CSF à des souris transgéniques
Alzheimer, n’ayant pas encore de symptômes, induit l’amélioration des fonctions cognitives, la
réduction d’Aβ et l’augmentation du nombre de microglies (315). Lorsqu’administré après le
développement de plaques amyloïdes, le traitement au M-CSF stabilise le déclin cognitif sans le
renverser (23,315). De plus, le M-CSF induit l’acidification des lysosomes, ce qui favorise la
phagocytose (352). Semblable au M-CSF, le G-CSF diminue l’accumulation et la déposition de
l’Aβ en activant les microglies et promouvant le recrutement des macrophages circulants (353). De
manière intéressante, le traitement des souris APPswe/PS1 avec t-PA induit des effets semblables
au M-CSF et G-CSF. Ainsi, t-PA constitue un traitement intéressant à développer. Pour ce faire, des
études additionnelles sur l’effet de t-PA sur les cellules exprimant LRP1, les péricytes et les
astrocytes, doivent être faites, puisque comprendre l’ensemble des mécanismes induits par rt-PA est
crucial. L’application de ce traitement chez des souris plus âgées, vers 6-7 mois, permettrait de
définir l’étendue de l’effet de t-PA sur la fonction cognitive plutôt que son effet préventif.
L’administration de l’Activase® rt-PA chez des souris plus vieilles permettrait également de
s’assurer qu’aucun effet néfaste n’est induit avec la perméabilisation de la BBB associé à l’âge
(241). En effet, avec la rupture de la BBB, l’incidence d’hémorrhagies cérébrales risque
d’augmenter (219–221). Le cas échéant, le traitement des patients par le t-PA serait restreint ceux
sans trouble vasculaire.
89
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Annexe – Figures supplémentaires
Chapitre 2
Figure. 2.1. Motricity behavior in APPswe/PS1 after severe chronic cerebral hypoperfusion (SCCH).
Motricity performance was assessed by open field test (A) and asymmetry cylinder test (B) which
respectively defined motor capacity and lateral asymmetry. Overall, both groups performed similarly
suggesting that SCCH do not induce motor impairment.
110
Figure 2.2. SCCH does not alter blood-brain barrier tightness. BBB leakage is shown by
immunocytochemistry (A,B). Claudin V level is quantified by Western blot (C) and rationalised with β-
actin’s expression. SCCH neither induces IgG (A) and albumin’s (B) extravasation, nor changes of claudin V
expression (C). Control demonstrates IgG (A) and albumin (B) extravasation 48 hours after stroke. Images
were acquired with 1X objective. Scale bar = 1mm.
Figure 2.3. Absence of infiltred monocytes after SCCH. Monocyte (CD45+) infiltration is verified by
immunocytochemistry. Both, sham and SCCH, groups do not have monocyte infiltration. Control show
monocytes infiltration in the lesion region 48 hours after stroke. Images were acquired with 1X and 40X
objectives. Scale bar = 1mm (1X objective) ; Scale bar = 75µm (40X objective).
111
Figure 2.4. SCCH seems to atrophy CA3 without neuronal death. Neuronal death and structural changes
in hippocampus are respectively confirmed by FJB (A) and thionin staining (B). Control presented neuronal
death at the lesion site 48 hours later, whereas sham and SCCH groups do not (A). SCCH animals seem (P =
0.0739) to develop CA3 region’s atrophy, without alteration of CA1 and CA2 region and, dentelate gyrus (B).
Data are means ± SEM. Data are analysed by standard two-tailed unpaired t-test’s. FJB staining images were
acquired with 1.25X and 20X objectives. Thionin staining images were acquired with 1X and 20X objectives.
Scale bar = 1mm (1.25X objective); Scale bar = 500µm (1X objective) ; Scale bar = 75µm (20X objective).
112
Chapitre 3
Figure. 3.1. BBB tightness is preserved after Activase® rt-PA administration. Microphotograph (a) and
immunohistochemical analysis (b,c) examining the purity of isolated cerebral MVs, and BBB permeability to
blood-borne molecules in the brain of APPswe/PS1 mice. The bright-field microphotograph outlines the
purity of isolated brain MVs (a). The immunohistochemical analysis shows no albumin (b) and IgG (c)
extravasation staining in the brain parenchyma of APPswe/PS1 mice 10 weeks after Activase® rt-PA weekly
systemic administration. Images were acquired with a 40X objective. Scale bar = 50 µm (a), 25 µm (b,c).
113
Figure. 3.2. BBB integrity is preserved after Activase® rt-PA administration. Western blot analysis,
using brain capillary extracts from wildtype mice and examining the expression levels of proteins involved in
BBB physical and functional proprieties, 3 (a,c,e) and 24 hours (b,d,f) after a single systemic Activase® rt-
PA administration. The protein expression levels of Occludin (a,b), Claudin 5 (c,d), and ABCB1 (e,f), do not
change. Optical densities were corrected with β-actin levels. Data are means ± SEM (n = 5-6).
114
Figure. 3.3. Endothelial transporters involved in Aβ transport across the BBB are not affected
following Activase® rt-PA administration. Western blot analysis, using brain MVs extracts from wildtype
mice and examining the expression levels of endothelial transporters involved in Aβ transport across the
BBB, 3 (a,c) and 24 hours (b,d) after a single systemic Activase® rt-PA administration. The protein
expression levels of LRP1 (a,b) and RAGE (c,d), do not change. Optical densities were corrected with β-actin
levels. Data are means ± SEM (n = 5-6).
115
Figure. 3.4. Activase® rt-PA regimen does not modulate the brain levels of synaptophysin.
Immunofluorescence staining (a) and western blot analysis (b) examining synaptophysin levels in the brain of
of APPswe/PS1 mice. Immunofluorescence staining shows that Activase® rt-PA treatment does not change
the brain levels of synaptophysin in treated APPswe/PS1 mice (a). Western blot analysis shows that
synaptophysin levels are decreased in the brain of APPswe/PS1 mice compared to wildtype littermate (b).
However, Activase® rt-PA treatment does not change synaptophysin levels in the brain of APPswe/PS1
treated mice (b). WT: wildtype. Data are means ± SEM (n = 6-7). **** P < 0.0001 compared with saline
treated group. Immunofluorescent images were acquired with a 4X objective. Scale bar = 500 µm.
116
Figure. 3.5. Acute Activase® rt-PA administration modulates total monocyte frequency in the blood of
APPswe/PS1 mice. Flow cytometry analysis was performed to examine total monocyte population frequency
and subset frequencies in the blood of APPswe/PS1 mice. Activase® rt-PA increases total monocyte
frequency in leukocytes (CD45+ cells) in the blood 3 hours after injection (a). However, Activase® rt-PA
does not modulate Ly6CHigh monocyte subset frequency (b) and Ly6CLow subset frequency (c) in the blood 3
hours after injection. Data are means ± SEM (n = 7-8 animals per group). * P < 0.05 compared with saline
treated group.
Figure. 3.6. Chronic Activase® rt-PA administration does not trigger a sustained inflammation in the
brain of chimeric APPswe/PS1 mice. In situ hybridization examining IkBα expression, an indicator of NF-
kB activity, following Activase® rt-PA administration. The acute administration of rt-PA does not induce
NF-kB activation (a), which is assessed by the expression levels of IkBα gene transcript. Data are means ±
SEM (n = 4-5). * P < 0.05 compared with saline treated group. Brain sections of mice systemically injected
with LPS were used as positive controls for NF-kB activation. Images were acquired with a 4X objective.
Scale bar = 250 µm.
117
Chapitre 4
Figure 4.1. Changement de la déposition vasculaire de l’amyloïde. L’amyloïde vasculaire a été marquée
par immunohistochimie à la Thioflavine 0,1%, puis analysé à l’aide du programme stéréo Investigateur. À la
suite de la SCCH, le nombre de plaques vasculaires diminue significativement comparé au contrôle, sans que
l’aire occupée par les plaques ne soit modulé. Les valeurs représentent la moyenne ± SEM (n = 6-8, 4 sections
du cerveau analysés par animal). L’amyloïde vasculaire** P < 0.001 comparé au groupe contrôle (SHAM).