Alzheimer’s disease (AD), the most common form of age-related dementia, is characterized by a progressive degeneration of the central nervous system (CNS) that leads to a gradual decline of cognitive functions and memory loss. Neuropathological hallmarks of AD include extracellular beta-amyloid plaques, derived from the altered processing of amyloid precursor protein (APP), neurofibrillary tangles (NFTs, intraneuronal aggregates of hyperphosphorylated and misfolded tau), dystrophic neurites, neuronal loss and glial activation. According to the “Amyloid cascade hypothesis” - the most validated theory in the field of AD for the past few decades - neuroinflammation was assumed to occur only in the late stages of the disease, being considered as a mere secondary response to Abeta-induced pathophysiological events. Recently, new preclinical, epidemiological and genetic studies have demonstrated a much earlier involvement of immune system-related actions, leading to a reassessment of the role of the principal innate immune entities of the brain, that are microglia cells. Since there is still no cure for AD, these studies motivated the design of innovative therapeutic strategies aiming at slowing down degenerative processes by targeting microglia cells, in virtue of their main recognized role in orchestrating neuroinflammatory process in neurodegenerative diseases, including AD. Mesenchymal stem cells (MSCs) are adult multipotent stem cells that over the last decades have been demonstrated to show improvement in various model of neurodegenerative pathologies, thanks to their paracrine ability that is largely dependent on the secretion of extracellular vesicles (EVs). EVs - membrane bound entities known to be important players in intercellular communication - have emerged as mediator of multiple MSC beneficial effects, including immunomodulation. Particularly, the concept that intrinsic immunomoregulatory abilities of MSCs are strongly influenced and strengthened by the environment, has led the scientists to design and optimize culture conditions (preconditioning) in order to enhance the anti-inflammatory properties of these cells and of their derived EVs. The aim of this study is to investigate the ability of preconditioned human bone marrow MSC-derived EVs (p-MSC-EVs) to immunoregulate microglia function in vitro and in vivo AD context. In in vitro studies we tested two different preconditioning protocols in order to isolate a highly immunomodulant MSC phenotype. Cytokine p-MSC-EVs were shown to switch microglia, previously polarized through inflammatory challenge to the M1 cytotoxic state, toward an anti-inflammatory phenotype. When we delved into the EV immunomodulatory potential in a triple transgenic AD (3xTg AD) mouse model, we observed a strong dampening effect on microglia activation and prevention of dendritic spine loss in hippocampus, entorhinal and prefrontal cortices of EV treated animals compared to controls. This suggests that an EV-dependent neuroprotective effect could be achieved through the modulation of microglia activation in this model. In order to more selectively study the effect of EVs on microglia, we are taking advantage of a leech animal model (Hirudo verbana), because of its simple and well-characterized CNS structure (preliminary study). In conclusion, our results indicate that p-MSC-EVs may represent a possible therapeutic tool in AD by reducing chronic microglia activation and counteracting dendritic spine loss, which are traits typically observed both in AD transgenic animal models and patients.

La malattia di Alzheimer (AD), la forma più comune di demenza senile, è caratterizzata da una progressiva degenerazione del sistema nervoso centrale (SNC) che conduce ad un declino della funzione cognitiva e a perdita di memoria. I tratti caratteristici dell’AD includono placche extracellulari di beta amiloide (Abeta), grovigli neurofibrillari (NFTs), neuriti distrofici, morte neuronale e attivazione gliale. In base a quanto affermato dall’ “Ipotesi della cascata della Abeta” - la teoria più corroborata nell’ambito degli studi sull’AD negli ultimi decenni – la neuroinfiammazione veniva supposta come un processo caratterizzante solo le fasi tardive della malattia, e considerata come una mera risposta secondaria agli eventi patologici indotti dall’ A beta. Recentemente, nuovi studi preclinici, epidemiologici e genetici hanno dimostrato un coinvolgimento del sistema immunitario molto più precoce, comportando una rivalutazione del ruolo della principale cellula dell’immunità innata del cervello: la microglia. Dal momento che non vi è cura per l’AD, questi studi hanno motivato lo sviluppo di strategie terapeutiche con l’obiettivo di rallentare i processi degenerativi attraverso un’azione sulle cellule microgliali, in virtù del loro ruolo nell’orchestrare la neuroinfiammazione nelle malattie neurodegenerative. Le cellule staminali mesenchimali (MSC) sono cellule staminali multipotenti che negli ultimi decenni sono emerse per la loro capacità di migliorare il decorso patologico in diversi modelli di malattie neurodegenerative, grazie alla loro attività paracrina, che in gran parte dipende dal rilascio di vescicole extracellulari (EV). Le EV - strutture lipidiche importanti per la comunicazione intercellulare - si sono dimostrate essere mediatori di molti effetti benefici indotti dalle MSC, come l’immunomodulazione. Nello specifico, il concetto che le intrinseche abilità immunoregolatorie delle MSC sono fortemente potenziate dall’ambiente in cui si trovano, ha portato gli scienziati a ridisegnare e ad ottimizzare le condizioni di coltura (precondizionamento, P) di queste cellule, per potenziare le proprietà anti-infiammatorie delle stesse e delle EV da loro rilasciate. L’obiettivo di questo studio è quello di analizzare la capacità delle EV rilasciate da MSC umane precondizionate (p-MSC-EV) di immunoregolare la funzione della microglia in contesti in vitro ed in vivo di AD. Negli studi in vitro abbiamo testato due differenti protocolli di P al fine di isolare un fenotipo altamente immunomodulante di MSC. Le EV derivate da MSC precondizionate con citochine si sono dimostrate in grado di polarizzare il fenotipo microgliale, precedentemente indotto tramite stimoli infiammatori in uno stato funzionale citotossico, verso un fenotipo anti-infiammatorio. Quando abbiamo indagato il potenziale immunomodulatorio delle EV in un modello murino di AD (3xTg AD), abbiamo osservato un forte effetto attenuante sull’attivazione microgliale e sulla prevenzione della perdita di spine dendritiche nelll’ippocampo e nelle cortecce entorinali e prefrontali degli animali trattati con le EV rispetto ai controlli. Questo suggerisce che un effetto neuroprotettivo potrebbe essere ottenuto tramite la modulazione dell’attivazione microgliale in questo modello. Per studiare più selettivamente l’effetto delle EV sulla microglia, stiamo sfruttando un modello di sanguisuga (Hirudo verbana), grazie alla semplice e ben caratterizzata struttura del suo SNC (studio preliminare). In conclusione, i nostri risultati indicano che le p-MSC-EV potrebbero rappresentare uno strumento terapeutico nell’AD, attraverso la riduzione dell’attivazione microgliale e contrastando la perdita di spine dendritiche, tratti neuropatologici tipicamente presenti sia nei modelli transgenici di AD che nei pazienti.

(2019). A NEW HOPE FOR ALZHEIMER’S DISEASE FROM PRECONDITIONED BONE MARROW MESENCHYMAL STEM CELL-DERIVED EXTRACELLULAR VESICLES: ANALYSIS OF THE IMMUNOMODULATORY EFFECTS. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).

A NEW HOPE FOR ALZHEIMER’S DISEASE FROM PRECONDITIONED BONE MARROW MESENCHYMAL STEM CELL-DERIVED EXTRACELLULAR VESICLES: ANALYSIS OF THE IMMUNOMODULATORY EFFECTS

LOSURDO, MORRIS
2019

Abstract

Alzheimer’s disease (AD), the most common form of age-related dementia, is characterized by a progressive degeneration of the central nervous system (CNS) that leads to a gradual decline of cognitive functions and memory loss. Neuropathological hallmarks of AD include extracellular beta-amyloid plaques, derived from the altered processing of amyloid precursor protein (APP), neurofibrillary tangles (NFTs, intraneuronal aggregates of hyperphosphorylated and misfolded tau), dystrophic neurites, neuronal loss and glial activation. According to the “Amyloid cascade hypothesis” - the most validated theory in the field of AD for the past few decades - neuroinflammation was assumed to occur only in the late stages of the disease, being considered as a mere secondary response to Abeta-induced pathophysiological events. Recently, new preclinical, epidemiological and genetic studies have demonstrated a much earlier involvement of immune system-related actions, leading to a reassessment of the role of the principal innate immune entities of the brain, that are microglia cells. Since there is still no cure for AD, these studies motivated the design of innovative therapeutic strategies aiming at slowing down degenerative processes by targeting microglia cells, in virtue of their main recognized role in orchestrating neuroinflammatory process in neurodegenerative diseases, including AD. Mesenchymal stem cells (MSCs) are adult multipotent stem cells that over the last decades have been demonstrated to show improvement in various model of neurodegenerative pathologies, thanks to their paracrine ability that is largely dependent on the secretion of extracellular vesicles (EVs). EVs - membrane bound entities known to be important players in intercellular communication - have emerged as mediator of multiple MSC beneficial effects, including immunomodulation. Particularly, the concept that intrinsic immunomoregulatory abilities of MSCs are strongly influenced and strengthened by the environment, has led the scientists to design and optimize culture conditions (preconditioning) in order to enhance the anti-inflammatory properties of these cells and of their derived EVs. The aim of this study is to investigate the ability of preconditioned human bone marrow MSC-derived EVs (p-MSC-EVs) to immunoregulate microglia function in vitro and in vivo AD context. In in vitro studies we tested two different preconditioning protocols in order to isolate a highly immunomodulant MSC phenotype. Cytokine p-MSC-EVs were shown to switch microglia, previously polarized through inflammatory challenge to the M1 cytotoxic state, toward an anti-inflammatory phenotype. When we delved into the EV immunomodulatory potential in a triple transgenic AD (3xTg AD) mouse model, we observed a strong dampening effect on microglia activation and prevention of dendritic spine loss in hippocampus, entorhinal and prefrontal cortices of EV treated animals compared to controls. This suggests that an EV-dependent neuroprotective effect could be achieved through the modulation of microglia activation in this model. In order to more selectively study the effect of EVs on microglia, we are taking advantage of a leech animal model (Hirudo verbana), because of its simple and well-characterized CNS structure (preliminary study). In conclusion, our results indicate that p-MSC-EVs may represent a possible therapeutic tool in AD by reducing chronic microglia activation and counteracting dendritic spine loss, which are traits typically observed both in AD transgenic animal models and patients.
COCO, SILVIA
AD; Neuroinfiammazione; Microglia; MSC; EV
AD; Neuroinflammation; Microglia; MSC; EV
BIO/14 - FARMACOLOGIA
English
4-feb-2019
NEUROSCIENZE - 90R
31
2017/2018
open
(2019). A NEW HOPE FOR ALZHEIMER’S DISEASE FROM PRECONDITIONED BONE MARROW MESENCHYMAL STEM CELL-DERIVED EXTRACELLULAR VESICLES: ANALYSIS OF THE IMMUNOMODULATORY EFFECTS. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/241093
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