In recent years, the concept of neurovascular unit (NVU) has progressively been entered. The NVU comprises multiple types of brain cells, including endothelial cells (ECs), astrocytes, pericytes, neurons and microglia (MG). Each cell type contributes to the maintenance of the transport through the blood–brain barrier (BBB) and brain tissue homeostasis. NVU dysfunctions have been associated with several disorders of the central nervous system (CNS) and neurodegenerative diseases. As a result, demand for therapeutic tools able to modulate the NVU has been increased. We firstly investigated the inner mechanism of action of liposomes (mApoE-PA-LIP) functionalized with a peptide derived from the apolipoprotein-E receptor-binding domain (mApoE) for BBB targeting and with phosphatidic acid (PA) for amyloid-β (Aβ) binding, previously used to promote peptide removal across the BBB and its peripheral clearance in a mouse model of Alzheimer's disease (AD) (Balducci et al., 2014). In light of previous results, we assessed whether mApoE-PA-LIP impacted on intracellular calcium ([Ca2+]i) dynamics in cultured human cerebral microvascular ECs (hCMEC/D3), as an in vitro human BBB model, and in cultured astrocytes. mApoE-PA-LIP pre-treatment increased the ATP-evoked Ca2+ waves in both cell types, also under 0 [Ca2+]e conditions, thus indicating that this increase is mainly due to endogenous Ca2+ release and that metabotropic purinergic receptors (P2Y) are mainly involved. Considering that P2Y receptors represent important pharmacological targets to treat cognitive dysfunctions and their neuroprotective effects in neuroinflammatory processes, the enhancement of purinergic signalling provided by mApoE-PA-LIP could counteract Aβ-induced vasoconstriction and reduction in cerebral blood flow (Forcaia et al., 2021). Moreover, we performed electrophysiological recordings on mouse brain slices to assess whether mApoE-PA-LIP can also modulate the neuronal synaptic transmission. Indeed, mApoE-PA-LIP treatment increased spontaneous excitatory postsynaptic currents (sEPSCs) frequency, giving additional support to promote mApoE-PA-LIP as putative therapeutic tool for AD treatment. In recent years, human epidemiological and animal studies put in evidence how the CNS is emerging as an important target for adverse health effects of airborne pollutants (AP) (Costa et al., 2020), demonstrating also that AP-exposure may also induce synaptic plasticity impairment (Hajipour et al., 2020). Thus, taking into account that the inhalation of airborne particulate matter (PM) may contribute to the neurodegeneration onset and progression, we moved furthermore towards understanding the inner underlying mechanisms. In lights of this evidence, we investigated the direct effect of AP on hCMEC/D3, that are the first interface between blood and brain, and MG, the main effector of neuroinflammation, using a standard reference material of diesel exhaust particles (DEP), that is one of the main PM contributors. To study the PM impact on purinergic signalling, we compared untreated primary MG with those incubated with DEP (10 μg/cm2 for 24 hours). Our data showed that DEP-activated MG generated much smaller Ca2+ signals ATP-induced, revealing a significant suppression of the receptor-evoked Ca2+ signals, probably due to an increase in the resting [Ca2+]i associated with MG activation (Hoffmann et al., 2003) that in our experiments could be confirmed also from the morphological analysis that revealed ameboid MG phenotype. The same results have been obtained in hCMEC/D3, indicating that the direct exposure to DEP causes also ECs activation. The here outlined results relating to the DEP-induced effects on ECs and on MG stated that DEP causes NVU physiology alterations which, adding together, could lead to the lowering of individual threshold to the onset of neurodegeneration.

Negli ultimi anni il concetto di unità neurovascolare (NVU) è stato progressivamente definito. La NVU comprende diversi tipi di cellule cerebrali: le cellule endoteliali (CE), gli astrociti, i periciti, i neuroni e la microglia (MG). Ciascuno di essi contribuisce al mantenimento della barriera ematoencefalica (BEE) e all'omeostasi del cervello. Disfunzioni a livello della NVU sono stati associati a diversi disturbi del sistema nervoso centrale (SNC) e a malattie neurodegenerative. Di conseguenza, è aumentata la richiesta di strumenti terapeutici in grado di modulare la NVU. In primo luogo, abbiamo studiato il meccanismo d'azione di liposomi (mApoE-PA-LIP) funzionalizzati con un peptide dell'apolipoproteina-E (mApoE) che conferisce affinità alla BEE e con l’acido fosfatidico (PA) per il legame con la β-amiloide (Aβ), precedentemente utilizzati per promuovere la rimozione di Aβ attraverso la BEE in un modello murino della malattia di Alzheimer (AD) (Balducci et al., 2014). Abbiamo pertanto valutato l’impatto dei mApoE-PA-LIP sulle dinamiche di calcio intracellulare ([Ca2+]i) nelle CE microvascolari cerebrali umane in coltura (hCMEC/D3), come modello in vitro di BEE e negli astrociti in coltura. Il pre-trattamento con mApoE-PA-LIP ha aumentato la [Ca2+]i indotta dall'ATP in entrambi i tipi cellulari, anche in condizioni 0 [Ca2+]e, indicando così che questo aumento è principalmente dovuto al rilascio endogeno di Ca2+ e al coinvolgimento dei recettori metabotropici purinergici (P2Y). Considerando che essi rappresentano un importante bersaglio farmacologico per il trattamento delle disfunzioni cognitive e considerando il loro effetto protettivo nei processi neuroinfiammatori, il potenziamento della trasmissione purinergica potrebbe contrastare la vasocostrizione indotta da Aβ e la riduzione del flusso sanguigno cerebrale (Forcaia et al., 2021). Inoltre, abbiamo eseguito registrazioni elettrofisiologiche su fettine di cervello di topo per valutare se i mApoE-PA-LIP potessero modulare anche la trasmissione sinaptica. In effetti, il trattamento con i mApoE-PA-LIP ha aumentato la frequenza delle correnti postsinaptiche eccitatorie spontanee (sEPSC), fornendo ulteriore supporto nel promuovere i mApoE-PA-LIP come possibile strumento terapeutico per il trattamento dell'AD. Negli ultimi anni, studi animali e sull’uomo hanno evidenziato che il SNC è un importante bersaglio degli effetti nocivi degli inquinanti atmosferici (Costa et al., 2020), dimostrando anche che l'esposizione ad essi può indurre anche alterazioni della plasticità sinaptica (Hajipour et al., 2020). Pertanto, considerando che l'inalazione di particolato (PM) può contribuire all'insorgenza e alla progressione della neurodegenerazione, ci siamo dedicati alla comprensione dei meccanismi sottesi. Abbiamo così studiato l'effetto diretto del PM sulle hCMEC/D3, che rappresentano la prima interfaccia tra sangue e cervello e la MG, il principale effettore della neuroinfiammazione, utilizzando un materiale di riferimento standard di particelle di scarico diesel (DEP), uno dei principali componenti del PM. Per studiare l'impatto sulla trasmissione purinergica, abbiamo confrontato la MG primaria con quella esposta a DEP (10 µg/cm2 per 24 ore). I nostri dati hanno indicato che la MG trattata mostra una significativa soppressione dei segnali di Ca2+ indotti dall'ATP, probabilmente a causa di un aumento del [Ca2+]i allo stato basale associato alla sua attivazione (Hoffmann et al., 2003), che nei nostri esperimenti potrebbe essere confermato anche dall'analisi morfologica (MG con fenotipo ameboide). Gli stessi risultati sono stati ottenuti nelle hCMEC/D3, indicando che l'esposizione diretta al DEP potrebbe provocare anche l'attivazione delle CE. Questi risultati indicano che il DEP provoca alterazioni fisiologiche della NVU che, sommandosi, potrebbero portare all'abbassamento della soglia individuale all'insorgenza di neurodegenerazione.

(2023). THE JANUS FACES OF THE NANOPARTICLES AT THE NEUROVASCULAR UNIT: A DOUBLE-EDGED SWORD IN NEURODEGENERATION. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2023).

THE JANUS FACES OF THE NANOPARTICLES AT THE NEUROVASCULAR UNIT: A DOUBLE-EDGED SWORD IN NEURODEGENERATION

TERRIBILE, GIULIA
2023

Abstract

In recent years, the concept of neurovascular unit (NVU) has progressively been entered. The NVU comprises multiple types of brain cells, including endothelial cells (ECs), astrocytes, pericytes, neurons and microglia (MG). Each cell type contributes to the maintenance of the transport through the blood–brain barrier (BBB) and brain tissue homeostasis. NVU dysfunctions have been associated with several disorders of the central nervous system (CNS) and neurodegenerative diseases. As a result, demand for therapeutic tools able to modulate the NVU has been increased. We firstly investigated the inner mechanism of action of liposomes (mApoE-PA-LIP) functionalized with a peptide derived from the apolipoprotein-E receptor-binding domain (mApoE) for BBB targeting and with phosphatidic acid (PA) for amyloid-β (Aβ) binding, previously used to promote peptide removal across the BBB and its peripheral clearance in a mouse model of Alzheimer's disease (AD) (Balducci et al., 2014). In light of previous results, we assessed whether mApoE-PA-LIP impacted on intracellular calcium ([Ca2+]i) dynamics in cultured human cerebral microvascular ECs (hCMEC/D3), as an in vitro human BBB model, and in cultured astrocytes. mApoE-PA-LIP pre-treatment increased the ATP-evoked Ca2+ waves in both cell types, also under 0 [Ca2+]e conditions, thus indicating that this increase is mainly due to endogenous Ca2+ release and that metabotropic purinergic receptors (P2Y) are mainly involved. Considering that P2Y receptors represent important pharmacological targets to treat cognitive dysfunctions and their neuroprotective effects in neuroinflammatory processes, the enhancement of purinergic signalling provided by mApoE-PA-LIP could counteract Aβ-induced vasoconstriction and reduction in cerebral blood flow (Forcaia et al., 2021). Moreover, we performed electrophysiological recordings on mouse brain slices to assess whether mApoE-PA-LIP can also modulate the neuronal synaptic transmission. Indeed, mApoE-PA-LIP treatment increased spontaneous excitatory postsynaptic currents (sEPSCs) frequency, giving additional support to promote mApoE-PA-LIP as putative therapeutic tool for AD treatment. In recent years, human epidemiological and animal studies put in evidence how the CNS is emerging as an important target for adverse health effects of airborne pollutants (AP) (Costa et al., 2020), demonstrating also that AP-exposure may also induce synaptic plasticity impairment (Hajipour et al., 2020). Thus, taking into account that the inhalation of airborne particulate matter (PM) may contribute to the neurodegeneration onset and progression, we moved furthermore towards understanding the inner underlying mechanisms. In lights of this evidence, we investigated the direct effect of AP on hCMEC/D3, that are the first interface between blood and brain, and MG, the main effector of neuroinflammation, using a standard reference material of diesel exhaust particles (DEP), that is one of the main PM contributors. To study the PM impact on purinergic signalling, we compared untreated primary MG with those incubated with DEP (10 μg/cm2 for 24 hours). Our data showed that DEP-activated MG generated much smaller Ca2+ signals ATP-induced, revealing a significant suppression of the receptor-evoked Ca2+ signals, probably due to an increase in the resting [Ca2+]i associated with MG activation (Hoffmann et al., 2003) that in our experiments could be confirmed also from the morphological analysis that revealed ameboid MG phenotype. The same results have been obtained in hCMEC/D3, indicating that the direct exposure to DEP causes also ECs activation. The here outlined results relating to the DEP-induced effects on ECs and on MG stated that DEP causes NVU physiology alterations which, adding together, could lead to the lowering of individual threshold to the onset of neurodegeneration.
SANCINI, GIULIO ALFREDO
Unita neurovascolare; Neurodegenerazione; Nanoparticelle; Nanomedicina; Inquinanti dell'aria
Neurovascular unit; Neurodegeneration; Nanoparticles; Nanomedicine; Air pollutants
BIO/09 - FISIOLOGIA
English
30-gen-2023
NEUROSCIENZE
35
2021/2022
open
(2023). THE JANUS FACES OF THE NANOPARTICLES AT THE NEUROVASCULAR UNIT: A DOUBLE-EDGED SWORD IN NEURODEGENERATION. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2023).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/403042
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