Particulate matter (PM) is a complex mixture of solid and liquid particles suspended in the air, and this suspension could be formed by a variety of particles of different size and composition depending on their origin (Marconi, 2003). Among the different fractions, ultrafine particles (UFPs) are thought to have the greatest health effects because of their characteristics. UFPs derive primarily from combustion processes in urban settings (Cassee et al., 2013) and, in the Lombardy Region, solid biomass burning (BC) for residential heating and diesel (DEP) combustion used for private and public transport represent their major sources (Longhin et al., 2016). Interestingly, emerging evidences from different studies suggest that neurological diseases, such as AD, PD and stroke, may be strongly associated with ambient PM (Genc at al., 2012). It has been demonstrated that continuous exposure to significant levels of airborne PM may result in the direct translocation of pollutants to different extra pulmonary sites, including central nervous system (CNS), or trigger the release of soluble inflammatory mediators from primary entry organs or secondary deposition sites (Genc at al., 2012). Systemic inflammation could activate cerebral endothelial cells, alter BBB integrity, or trigger signalling cascades that lead to the activation of MAPK and NFκB pathways (Calderón-Garcidueñas et al., 2008). Notably, post-mortem examinations of adult humans resident in highly polluted urban areas exhibited significantly higher brain COX-2 expression and accumulation of Aβ42 when compared to subjects living in cities with low pollution levels (Calderón-Garcidueñas et al., 2004). Therefore, the aim of this project was to evaluate the detrimental effect of UFPs exposure, regarding oxidative stress and inflammation, on in-vitro and in-vivo models of CNS. Moreover, this work meant to investigate the possible physiopathological correlation between these two mechanisms and AD neurodegeneration. First, we tested the effect of DEP administration on C6 glioma cells, which have properties of both astrocytes and oligodendrocytes. In fact, glial cells are now recognized as active players in the regulation of synaptic function, neural repair, and CNS immunity (Lee and MacLean, 2015), and astrocytes have been recently linked to neuroinflammatory and neurotoxic pathways induced by PM exposure (Li et al., 2016). We demonstrated that DEP treatment at sub-lethal concentrations induced oxidative stress in glial cells, while inflammation was not involved. Moreover, we found that C6 glioma cells activate anti-oxidant pathways to contrast the oxidative status induced by DEP treatment and that the MEK-ERK1-2 pathway seems important in regulating these anti-oxidant strategies. Afterwards, we selected HT22 nerve cell line as a neuronal in-vitro model to study the effect of direct DEP administration. We demonstrated that DEP treatment at sub-lethal concentrations induced oxidative stress and inflammation in neuronal cells, supporting the idea that neurons are more sensitive to DEP administration than glial cells. Moreover, we extended the analysis of DEP detrimental effects and we found lipid reshaping and alteration of APP and BACE1 protein levels in HT22 cells. Finally, we exposed male BALB/c mice to single and repeated Intratracheal instillation of BC and DEP by means of a MicroSprayer® Aerosolizer system. This analysis confirmed the inflammatory and oxidative potential of BC and DEP exposure on mouse brain, which was accompanied by induction of PAHs metabolism and alteration of APP processing after sub-acute exposure. In conclusion, these findings may contribute to the knowledge of the interplay between PM exposure, the chronic oxidative stress and inflammation generation and the development of neurodegenerative diseases.

Il particolato atmosferico (PM) è una miscela complessa di particelle solide e liquide sospese nell'atmosfera e questa sospensione può essere formata da una varietà di particelle di dimensione e composizione diversa a seconda della loro provenienza (Marconi, 2003). Tra le diverse frazioni di PM, si ritiene che le particelle ultrafini (UFP) provochino i maggiori effetti sulla salute a causa delle loro caratteristiche. Le UFP derivano principalmente da processi di combustione in ambienti urbani (Cassee et al., 2013) e, in Regione Lombardia, la combustione di biomassa solida (BC) per il riscaldamento residenziale e quella di diesel (DEP) per il trasporto pubblico e privato ne rappresentano le fonti principali (Longhin et al., 2016 ). Evidenze emergenti provenienti da diversi studi suggeriscono che le malattie neurologiche possono essere fortemente associate al PM ambientale (Genc at al., 2012). È stato dimostrato che l'esposizione continua a livelli significativi di PM può comportare la traslocazione diretta degli inquinanti a diversi siti extra-polmonari, compreso il sistema nervoso centrale (CNS), o innescare il rilascio di mediatori solubili dell’infiammazione dagli organi di ingresso primari o dai siti di deposizione secondaria (Genc at al., 2012). L’infiammazione sistemica potrebbe poi attivare le cellule endoteliali cerebrali, alterare l'integrità della BBB, o innescare processi che portano all’attivazione delle vie di segnalazione di MAPK e NFκB (Calderón-Garcidueñas et al., 2008). In particolare, esami post-mortem di adulti residenti in aree urbane altamente inquinate mostrano livelli cerebrali di COX-2 e accumulo di Aβ42 significativamente più alti rispetto a soggetti che vivono in città a bassi livelli di inquinamento (Calderón-Garcidueñas et al., 2004). Pertanto, l'obiettivo di questo progetto è stato quello di valutare su modelli in-vitro e in-vivo di CNS l'effetto dannoso dell'esposizione alle UFP e la sua possibile correlazione fisiopatologica con la neurodegenerazione tipica della malattia di Alzheimer (AD). In primo luogo, abbiamo testato l'effetto della somministrazione di DEP su cellule gliali, che attualmente sono riconosciute come soggetti attivi nella regolazione della funzione sinaptica, nella riparazione neurale e nell’immunità del CNS (Lee e MacLean, 2015) e sono state recentemente collegate a vie neuroinfiammatorie e neurotossiche indotte dall’esposizione al PM (Li et al., 2016). Abbiamo dimostrato che dosi sub-letali di DEP inducono stress ossidativo in cellule C6 glioma, mentre l'infiammazione non è coinvolta. Inoltre, abbiamo dimostrato che le cellule C6 glioma attivano vie antiossidanti per contrastare lo stato ossidativo indotto dal DEP e che la via MEK-ERK1-2 sembra importante nel regolare tali strategie. Successivamente, abbiamo dimostrato concentrazioni sub-letali di DEP inducono stress ossidativo e infiammazione in cellule neuronali HT22, supportando l’ipotesi che i neuroni siano più sensibili alla somministrazione rispetto alle cellule gliali. Inoltre, in seguito al trattamento abbiamo osservato una modulazione dei lipidi e un'alterazione dei livelli di espressione di APP e BACE1. Infine, abbiamo sottoposto topi BALB/c ad instillazione intratracheale singola e ripetuta con BC e DEP, attraverso il sistema MicroSprayer® Aerosolizer. Questa analisi ha confermato il potenziale infiammatorio e ossidativo di BC e DEP sul cervello dei topi, accompagnato da induzione del metabolismo degli idrocarburi policiclici aromatici e alterazione del processamento di APP dopo esposizione ripetuta. Per concludere, questi risultati contribuiscono a chiarire la relazione esistente tra esposizione al PM, generazione cronica di stress ossidativo e infiammazione e sviluppo di malattie neurodegenerative.

(2017). Air pollution in neurodegeneration: in-vitro and in-vivo models. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).

Air pollution in neurodegeneration: in-vitro and in-vivo models

MILANI, CHIARA
2017

Abstract

Particulate matter (PM) is a complex mixture of solid and liquid particles suspended in the air, and this suspension could be formed by a variety of particles of different size and composition depending on their origin (Marconi, 2003). Among the different fractions, ultrafine particles (UFPs) are thought to have the greatest health effects because of their characteristics. UFPs derive primarily from combustion processes in urban settings (Cassee et al., 2013) and, in the Lombardy Region, solid biomass burning (BC) for residential heating and diesel (DEP) combustion used for private and public transport represent their major sources (Longhin et al., 2016). Interestingly, emerging evidences from different studies suggest that neurological diseases, such as AD, PD and stroke, may be strongly associated with ambient PM (Genc at al., 2012). It has been demonstrated that continuous exposure to significant levels of airborne PM may result in the direct translocation of pollutants to different extra pulmonary sites, including central nervous system (CNS), or trigger the release of soluble inflammatory mediators from primary entry organs or secondary deposition sites (Genc at al., 2012). Systemic inflammation could activate cerebral endothelial cells, alter BBB integrity, or trigger signalling cascades that lead to the activation of MAPK and NFκB pathways (Calderón-Garcidueñas et al., 2008). Notably, post-mortem examinations of adult humans resident in highly polluted urban areas exhibited significantly higher brain COX-2 expression and accumulation of Aβ42 when compared to subjects living in cities with low pollution levels (Calderón-Garcidueñas et al., 2004). Therefore, the aim of this project was to evaluate the detrimental effect of UFPs exposure, regarding oxidative stress and inflammation, on in-vitro and in-vivo models of CNS. Moreover, this work meant to investigate the possible physiopathological correlation between these two mechanisms and AD neurodegeneration. First, we tested the effect of DEP administration on C6 glioma cells, which have properties of both astrocytes and oligodendrocytes. In fact, glial cells are now recognized as active players in the regulation of synaptic function, neural repair, and CNS immunity (Lee and MacLean, 2015), and astrocytes have been recently linked to neuroinflammatory and neurotoxic pathways induced by PM exposure (Li et al., 2016). We demonstrated that DEP treatment at sub-lethal concentrations induced oxidative stress in glial cells, while inflammation was not involved. Moreover, we found that C6 glioma cells activate anti-oxidant pathways to contrast the oxidative status induced by DEP treatment and that the MEK-ERK1-2 pathway seems important in regulating these anti-oxidant strategies. Afterwards, we selected HT22 nerve cell line as a neuronal in-vitro model to study the effect of direct DEP administration. We demonstrated that DEP treatment at sub-lethal concentrations induced oxidative stress and inflammation in neuronal cells, supporting the idea that neurons are more sensitive to DEP administration than glial cells. Moreover, we extended the analysis of DEP detrimental effects and we found lipid reshaping and alteration of APP and BACE1 protein levels in HT22 cells. Finally, we exposed male BALB/c mice to single and repeated Intratracheal instillation of BC and DEP by means of a MicroSprayer® Aerosolizer system. This analysis confirmed the inflammatory and oxidative potential of BC and DEP exposure on mouse brain, which was accompanied by induction of PAHs metabolism and alteration of APP processing after sub-acute exposure. In conclusion, these findings may contribute to the knowledge of the interplay between PM exposure, the chronic oxidative stress and inflammation generation and the development of neurodegenerative diseases.
PALESTINI, PAOLA NOVERINA ADA
BULBARELLI, ALESSANDRA
Diesel,; biomass,; oxidative; stress,; inflammation
Diesel,; biomass,; oxidative; stress,; inflammation
BIO/16 - ANATOMIA UMANA
English
3-apr-2017
NEUROSCIENZE - 90R
29
2015/2016
open
(2017). Air pollution in neurodegeneration: in-vitro and in-vivo models. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/158159
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