Air pollution (AP) is becoming the prominent environmental risk factor that affects public health and it is a mixture of several components, including ambient particulate matter (PM). One of the main contributors to PM is represented by traffic-related AP, mostly ascribed to Diesel Exhaust Particles (DEP), whose sizes contribute to their biological effects. The smaller ones can penetrate deep into the lungs and produce a systemic inflammation that can reach the Blood Brain Barrier (BBB), causing its structural damage and functional alterations. The BBB is centrally positioned in the Neurovascular Unit (NVU), which is a relatively recent concept in neuroscience that broadly describes the relationship between cerebral blood vessels and brain cells, including neurons, astrocytes, ECs, pericytes and microglia. Each component is intimately and reciprocally linked to each other, establishing an anatomical and functional whole, which results in a highly efficient system that preserve the homeostasis and the functionality of the brain microenvironment. In recent years, human epidemiological and animal studies show how the Central Nervous System is emerging as an important target for adverse health effects of AP, where they may be strongly associated with neurodegenerative disorders. Evidence supports AP exposure as a heavy risk for cognitive decline and the development of Alzheimer’s disease (AD). Moreover, different studies demonstrate how exposure to environmental AP induces synaptic plasticity impairment, both directly and indirectly. Since the topic is of fundamental importance and there are still very few clarifications, the race to gain in-depth knowledge is extremely urgent. Thus, we investigated how AP invalidates the mechanisms on which multiple neurotransmission signals depends, whose function is at the base of memory process. We exposed mice brain slices and in vitro NVU cells to DEP, a standard reference material (SRM), and we performed electrophysiological experiments through the Whole-Cell Patch-Clamp and Calcium Imaging technique on cortical pyramidal neurons and NVU cells, such as hCMEC/D3, a BBB model, and astrocytes, that indirectly regulate synaptic neurotransmission. Results show that DEP induce a decrease of neuronal sEPSCs/sIPSCs frequency and kinetic alterations, with the involvement of glutamatergic and GABAergic receptors. Moreover, DEP produce modifications on pre-synaptic mechanisms that modulate neurotransmitter release. Furthermore, DEP cause alterations of the Ca2+ dynamics in both hCMEC/D3 and astrocytes, affecting the overall NVU functionality and, consequently, the neurocommunication. As a result, demand for new therapeutic tools has been increased. Hereafter, our study is based on preliminary results that highlight the effects of multifunctional liposomes (mApoE-PA-LIP) as a putative therapeutic tool for AD treatment. The mApoE-PA-LIP can enhance NVU Ca2+ dynamics and ameliorate memory impairment of an AD mouse model. Thus, we tested the direct and indirect effect of mApoE-PA-LIP on synaptic transmission before and after DEP exposure. The Whole-Cell Patch-Clamp and the Calcium Imaging technique were performed with mApoE-PA-LIP perfusion on cortical pyramidal neurons of mice brain slices and on in vitro NVU cells. Results show that mApoE-PA-LIP can modulate an increase of sEPSCs/sIPSCs frequency both before and after DEP exposure, with the involvement of glutamatergic receptors. Moreover, mApoE-PA-LIP can induce a strong modulation of sEPSCs kinetics and of pre-synaptic neurotransmitter release. Furthermore, mApoE-PA-LIP can produce a recovery in Ca2+ dynamics of hCMEC/D3 after DEP exposure, but not in astrocytes. In conclusion, data suggest that AP produces critical direct and indirect alterations on physiological mechanisms of neurocommunication linked to AD development, for which mApoE-PA-LIP could be promoted as a strategy to counteract neurotransmission impairment.
L’inquinamento atmosferico (AP) sta diventando il principale fattore di rischio ambientale che incide sulla salute pubblica ed è una miscela di diversi componenti, tra cui il particolato ambientale (PM). Tra i principali componenti del PM vi è l’AP legato al traffico, per lo più attribuito alle Diesel Exhaust Particles (DEP). Le nanoparticelle penetrano producono un'infiammazione sistemica che raggiunge la barriera emato-encefalica (BBB), causando danni strutturali e alterazioni funzionali. La BBB è posizionata centralmente nell'unità neurovascolare (NVU), un concetto relativamente recente nelle neuroscienze che descrive la relazione tra i vasi sanguigni e le cellule cerebrali, inclusi neuroni, astrociti, EC, periciti e microglia. Ogni componente è intimamente e reciprocamente legato l’uno all’altro, costituendo un insieme anatomico e funzionale, che si traduce in un sistema preservante l'omeostasi e la funzionalità del microambiente cerebrale. Negli ultimi anni, diversi studi mostrano come il Sistema Nervoso Centrale sia un obiettivo importante per gli effetti avversi sulla salute causati dall’AP, i quali sono associati alle malattie neurodegenerative. Le evidenze scientifiche supportano l’esposizione all’AP come un forte rischio di sviluppo della malattia di Alzheimer (AD) e dimostrano che questa esposizione induce un’alterazione della plasticità sinaptica, sia direttamente che indirettamente. Poiché l’argomento è di fondamentale importanza e le conoscenze al riguardo sono ancora esigue, l’approfondimento di questo tema risulta quanto mai urgente. Pertanto, abbiamo studiato i meccanismi attraverso cui l'AP invalida la neurotrasmissione. Abbiamo esposto fettine di cervello di topo e cellule della NVU in vitro al DEP, un materiale di riferimento standard (SRM), e abbiamo eseguito esperimenti elettrofisiologici attraverso la tecnica di Whole-Cell Patch-Clamp e Calcium Imaging su neuroni corticali piramidali e cellule della NVU, come hCMEC/D3, un modello di BBB, e astrociti, che regolano indirettamente la trasmissione sinaptica. Il DEP causa una diminuzione della frequenza delle sEPSC/sIPSC neuronali, alterazioni cinetiche con il coinvolgimento dei recettori glutammatergici e GABAergici ed alterazioni dei meccanismi pre-sinaptici modulanti il rilascio dei neurotrasmettitori. Inoltre, il DEP modifica le dinamiche del Ca2+ sia nelle hCMEC/D3 che negli astrociti, influenzando la complessiva funzionalità della NVU e la neurocomunicazione. Di conseguenza, la necessità di nuovi strumenti terapeutici è aumentata. Di seguito, il nostro studio si basa su risultati preliminari che evidenziano gli effetti di liposomi multifunzionali (mApoE-PA-LIP) come presunto strumento terapeutico per il trattamento dell'AD, in quanto i mApoE-PA-LIP modulano le dinamiche del Ca2+ nella NVU e migliorano i deficit di memoria di un modello murino di AD. Pertanto, abbiamo testato l'effetto diretto e indiretto dei mApoE-PA-LIP sulla trasmissione sinaptica prima e dopo l'esposizione al DEP. Le tecniche di Whole-Cell Patch-Clamp e Calcium Imaging sono state eseguite perfondendo i mApoE-PA-LIP in neuroni piramidali corticali di fettine di cervello di topo e in cellule della NVU in vitro. I risultati mostrano che i mApoE-PA-LIP producono un aumento della frequenza delle sEPSC/sIPSC sia pre- che post-esposizione al DEP con il coinvolgimento dei recettori glutammatergici. I mApoE-PA-LIP inducono una forte modulazione della cinetica delle sEPSC e del rilascio pre-sinaptico dei neurotrasmettitori. Inoltre, i mApoE-PA-LIP producono un recovery delle dinamiche del Ca2+ nelle hCMEC/D3 dopo l'esposizione al DEP, ma non negli astrociti. In conclusione, i risultati suggeriscono che l’AP produca alterazioni critiche dirette e indirette sui meccanismi fisiologici legati allo sviluppo di AD, per le quali i mApoE-PA-LIP potrebbero essere promossi come strategia per contrastare le alterazioni della neurotrasmissione.
(2024). AIR POLLUTION NANOPARTICLES AND DOUBLE-FUNCTIONALIZED LIPOSOMES: THE CULPRITS AND RESCUERS OF THE IMPAIRED NEURONAL SYNAPTIC FUNCTION. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2024).
AIR POLLUTION NANOPARTICLES AND DOUBLE-FUNCTIONALIZED LIPOSOMES: THE CULPRITS AND RESCUERS OF THE IMPAIRED NEURONAL SYNAPTIC FUNCTION
DI GIROLAMO, SARA
2024
Abstract
Air pollution (AP) is becoming the prominent environmental risk factor that affects public health and it is a mixture of several components, including ambient particulate matter (PM). One of the main contributors to PM is represented by traffic-related AP, mostly ascribed to Diesel Exhaust Particles (DEP), whose sizes contribute to their biological effects. The smaller ones can penetrate deep into the lungs and produce a systemic inflammation that can reach the Blood Brain Barrier (BBB), causing its structural damage and functional alterations. The BBB is centrally positioned in the Neurovascular Unit (NVU), which is a relatively recent concept in neuroscience that broadly describes the relationship between cerebral blood vessels and brain cells, including neurons, astrocytes, ECs, pericytes and microglia. Each component is intimately and reciprocally linked to each other, establishing an anatomical and functional whole, which results in a highly efficient system that preserve the homeostasis and the functionality of the brain microenvironment. In recent years, human epidemiological and animal studies show how the Central Nervous System is emerging as an important target for adverse health effects of AP, where they may be strongly associated with neurodegenerative disorders. Evidence supports AP exposure as a heavy risk for cognitive decline and the development of Alzheimer’s disease (AD). Moreover, different studies demonstrate how exposure to environmental AP induces synaptic plasticity impairment, both directly and indirectly. Since the topic is of fundamental importance and there are still very few clarifications, the race to gain in-depth knowledge is extremely urgent. Thus, we investigated how AP invalidates the mechanisms on which multiple neurotransmission signals depends, whose function is at the base of memory process. We exposed mice brain slices and in vitro NVU cells to DEP, a standard reference material (SRM), and we performed electrophysiological experiments through the Whole-Cell Patch-Clamp and Calcium Imaging technique on cortical pyramidal neurons and NVU cells, such as hCMEC/D3, a BBB model, and astrocytes, that indirectly regulate synaptic neurotransmission. Results show that DEP induce a decrease of neuronal sEPSCs/sIPSCs frequency and kinetic alterations, with the involvement of glutamatergic and GABAergic receptors. Moreover, DEP produce modifications on pre-synaptic mechanisms that modulate neurotransmitter release. Furthermore, DEP cause alterations of the Ca2+ dynamics in both hCMEC/D3 and astrocytes, affecting the overall NVU functionality and, consequently, the neurocommunication. As a result, demand for new therapeutic tools has been increased. Hereafter, our study is based on preliminary results that highlight the effects of multifunctional liposomes (mApoE-PA-LIP) as a putative therapeutic tool for AD treatment. The mApoE-PA-LIP can enhance NVU Ca2+ dynamics and ameliorate memory impairment of an AD mouse model. Thus, we tested the direct and indirect effect of mApoE-PA-LIP on synaptic transmission before and after DEP exposure. The Whole-Cell Patch-Clamp and the Calcium Imaging technique were performed with mApoE-PA-LIP perfusion on cortical pyramidal neurons of mice brain slices and on in vitro NVU cells. Results show that mApoE-PA-LIP can modulate an increase of sEPSCs/sIPSCs frequency both before and after DEP exposure, with the involvement of glutamatergic receptors. Moreover, mApoE-PA-LIP can induce a strong modulation of sEPSCs kinetics and of pre-synaptic neurotransmitter release. Furthermore, mApoE-PA-LIP can produce a recovery in Ca2+ dynamics of hCMEC/D3 after DEP exposure, but not in astrocytes. In conclusion, data suggest that AP produces critical direct and indirect alterations on physiological mechanisms of neurocommunication linked to AD development, for which mApoE-PA-LIP could be promoted as a strategy to counteract neurotransmission impairment.File | Dimensione | Formato | |
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phd_unimib_765905.pdf
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Descrizione: Tesi di Di Girolamo Sara - 765905
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Doctoral thesis
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