Microglia-derived extracellular vesicles regulate the proliferation and differentiation of oligodendrocyte precursor cells
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1 University of Turin CNR Institute of Neuroscience Microglia-derived extracellular vesicles regulate the proliferation and differentiation of oligodendrocyte precursor cells Roberta Parolisi Turin, December 3, 2016
2 MICROGLIA & their roles Microglia (MG) are a type of small macrophage-like glial cells in the CNS. HOMEOSTASIS MG preserve homeostasis, and support neurons and glial cells M1 PATHOGENIC MG plays a role in disease progression, presenting antigens and secreting neurotoxic cytokines PROTECTIVE MG can suppress inflammation and cytodegeneration, and stimulate CNS repair, releasing trophic factors and anti-inflammatory cytokines Bogie et al., 2014
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4 MICROGLIA-DERIVED EXTRACELLULAR VESICLES The phenotype of MG influences the production and the functions of EVs. EXTRACELLULAR VESICLES (EVs) EVs are small membranous vesicles. from plasma membrane The EVs have been related to several physiological and pathological conditions. exocytosis of multivesicular bodies Verderio et al., 2012 Microglia-derived EVs reflect disease activity in MS patients
5 WE STUDIED WHETHER EVS RELEASED FROM MICROGLIA CAN BOOST OR BLOCK THE PROLIFERATION AND THE TERMINAL DIFFERENTIATION OF OPCs. Oligodedrocytes precursor cells Oligodedrocytes cells Neuron OL OPC
6 METHODS IN VITRO and IN VIVO ANALYSIS PROLIFERATION AND DIFFERENTIATION OF OPC Lombardi M., Fumagalli M. and Verderio C.
7 1.EFFECTS OF MICROGLIA-DERIVED EVs ON OPC PROLIFERATION M0 M1 unstimulated pro-inflammatory+mscs pro-inflammatory anti-inflammatory M0 UNSTIMULATED METHODS / Th1 M0 M1 to evaluate the action of microglial Evs on OPC proliferation OPC culture Lombardi M., Fumagalli M. and Verderio C.
8 1.EFFECTS OF MICROGLIA-DERIVED EVs ON OPC PROLIFERATION M0 M1 unstimulated pro-inflammatory+mscs pro-inflammatory anti-inflammatory Ctrl M1 M0 M1 inhibit OPC proliferation M0 M1 Pre-conditioning with MSCs abrogates the anti-proliferative effects of M1 Lombardi M., Fumagalli M. and Verderio C.
9 2.EFFECTS OF MICROGLIA-DERIVED EVs ON OPC DIFFERENTIATION M0 M1 unstimulated pro-inflammatory anti-inflammatory M0 UNSTIMOLATED METHODS M0 M1 to evaluate the action of microglial EVs on OPC differentiation ATP OPC culture Lombardi M., Fumagalli M. and Verderio C.
10 MBP DAPI MBP positive OPCs/ DAPI normalized value 2.EFFECTS OF MICROGLIA-DERIVED EVs ON OPC DIFFENTIATION M0 M1 unstimulated pro-inflammatory anti-inflammatory MBP/HOECHST Ctrl M1-EVs -EVs M0 M1 M B P p o s itiv e O P C s /D A P I N o rm a liz e d v a lu e *** *** **** M1 and promote OPC differentiation M0 0.0 Ctrl C T R L M 0 -M V s M0 M1 M 1 - M V s M 2 -M V s Lombardi M., Fumagalli M. and Verderio C.
11 3.EFFECTS OF MICROGLIA-DERIVED EVs ON MYELIN DEPOSITION M0 M1 unstimulated pro-inflammatory+mscs pro-inflammatory anti-inflammatory Ctrl M1 ** *** promote myelin deposition M0 M1 Lombardi M., Fumagalli M. and Verderio C.
12 4.EFFECTS OF EVs ON OPC PROLIFERATION (and differentiation) IN VIVO LYSOLECITHIN MOUSE MODEL OF FOCAL DEMYELINATION 2%LPC 7dpi NG2DAPI 7dpi MBP Gallyas 14dpi
13 NG2DAPI anti-inflammatory pro-inflammatory+mscs Liposomes Ctrl Liposomes/IL-4/Th1+MSCs BrdU 2pulses (2h) 7dpi Ctrl Ki67+ cells density ** Ng2+ cells density 250 *** IL4 LIPO Ctrl 0 IL4 LIPO Ctrl induce cell proliferation and an increased recruitment of NG2+ OPCs.
14 NG2BrdUDAPI anti-inflammatory pro-inflammatory+mscs Liposomes Ctrl Liposomes/IL-4/Th1+MSCs BrdU 2pulses (2h) 10dpi NG2BrdUDAPI NG2-BrdU+ NG2+BrdU+ Ng2 BrdU+ cells/ mm 2 Ng2 BrdU+ cells/ mm Ctrl With cronic administration of the new born NG2+ OPCs seem to be increased up to 10 days after lesion.
15 SUMMARY AMPLIFICATION DIFFERENTIATION M1 pro-inflammatory+mscs pro-inflammatory anti-inflammatory simplified system AMPLIFICATION DIFFERENTIATION +?? + multicellular environment EVs have a different effects in vivo and in vitro. In vivo, EVs could also affect other cell types, with secondary effects.
16 We are analysing the effects of EVs on differentiation to evaluate:? Hypothesis 1 this accumulation of OPCs pool is able to generate a greater number of mature cells (OLs) after differentiation, getting a massive myelination? Hypothesis 2 this accumulation of OPC pool is unable to differentiate and is blocked at this phase time
17 Laboratory of Neurobiology of brain plasticity Annalisa Buffo Enrica Boda CNR Institute of Neuroscience Claudia Verderio Marta Lombardi Martina Gabrielli University of Milan Maria Pia Abbracchio Marta Fumagalli Elisabetta Bonfanti
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19 qrt-pcr analysis to confirm microglia polarization TNFalfa COX2 inos M1 M1 M1
20 Annex-PE MICROGLIA-DERIVED EXTRACELLULAR VESICLES Ectosomes 10 5 Flow cytometry 3% 32% % IB4-FICT Nanosight size plots
21 Concentration (particles/ml) Quantification and size of EVs from M0, M1 and microglia CTR M1 particles secreted by 1x10^6 MG 1.20E E E E E E E+000 CTR M1 Particle Diameter (nm) NO changes in the size distribution of M1-EVs or -EVs but increased EV production from M1 but not microglia as compared to unstimulated cells
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23 IBA1DAPI
24 M0-EVs are able to act as chemoattractants for OPCs, similarly to -EVs overnight Evs are added to the culture medium of the outer well. OPCs are placed in the upper chamber Migratory cells pass through PET membrane. Non-migratory cells stay in the upper chamber % of migrated cells over control CTRL REST OPCs Free Medium PET membrane After removal of nonmigratory cells, migratory cells are stained and quantified Medium with EVs Staining solution Bar graph shows the percentage of migrated OPCs after an overnight exposure of EVs derived from REST or microglia with respect to control cells set to 100%. The number of migrated HOECHST + cells was counted in 60 optical fields at 20x magnification. Data are the mean±s.e.m. of cell count of 3 transwells/condition from one experiment.
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27 MULTIPLE STRATEGIES TO PROMOTE ENDOGENOUS REMYELINATION VIA THE ACTION OF EVS RELEASED FROM ACTIVATED MG From Chandran et al., 2008
28 ACUTE ADMINISTRATION
![-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3](/docs-images/95/125847673/images/29-2.jpg "-p-phenylene-bis-acrylamide dihydrochloride)")
29 GW4869 inhibit nsmase2 in vivo. GW4869-treated mice produce reduced amounts of exosomes. GW4869 (N,N -Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3 -p-phenylene-bis-acrylamide dihydrochloride) Nuclear Fas Red Luxol Fast Blue Intraperitoneal administration of GW4869 at 2 µg/g mouse body weight.
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SUPPLEMENTARY FIG. S2. Representative counting fields used in quantification of the in vitro neural differentiation of pattern of dnscs.
Supplementary Data SUPPLEMENTARY FIG. S1. Representative counting fields used in quantification of the in vitro neural differentiation of pattern of anpcs. A panel of lineage-specific markers were used
Supplementary Figure 1
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