One of the most complex challenges in clinical research is the development of experimental animal models able to reproduce the physiopathological features of human diseases as closely as possible. Regarding Parkinson's disease (PD), several models have been generated over the years: from toxicological ones (with MPTP, 6-OHDA, rotenone) (Schober, 2004; Sherer et al., 2003) to transgenic ones (Dawson et al., 2010; Masliah et al., 2000) and, more recently, models based on the prion-like behaviour of α-synuclein (α-Syn) (Cenci & Björklund, 2020). These are used not only to understand the mechanisms underlying the development and progression of the disease, but also to determine the effectiveness of a given therapeutic approach. Therefore, this thesis aims to characterize a new animal experimental model, based on the use of preformed α-synuclein (α-Syn) fibrils (PFFs) injected directly into the SN of C57BL mice. Once they reach the neurons, the PFFs trigger the nucleation process of the endogenous α-Syn, which leads to the formation of protein aggregates typical of PD. The results obtained show, firstly, the ability of the PFFs to cause the aggregation of endogenous α-Syn, both at the injection site and in the structures directly connected to it. The aggregation is already visible two weeks after the injection and remains constant over time. There is also a progressive increase in the neurodegeneration of dopaminergic neurons, a fundamental feature in a PD model. Finally, the fundamental parameter analysed was the neuroinflammation. Given that microglia is the most important cell type in the brain involved in the resolution of damage caused by pathologies, injuries, or infections, we decided to measured the microglial activation. By measuring the presence of microglial markers such as IBA1 and CD68, the presence of a strong microglial activation was confirmed at the earliest time points, which is followed by a decrease after four months. The second part of this project focuses specifically on the role of microglia with the view to develop an effective gene therapy approach based on the "microRNA detargeting" strategy. With this in mind, we generate a lentiviral vector which allows the transgene expression exclusively in the microglial cells. To do this, a particular sequence was used, called mirTAG, which is complementary to a specific microRNAs present in different cell types (neurons, astrocytes, oligodendrocytes, endothelium) but almost absent in the microglia. In this way, the lentiviral genome is preserved only in microglia where, therefore its expression is permitted. The construct was initially tested with green fluorescent protein (GFP) both in vitro and in vivo in order to confirm the specificity of expression. Then, the gene of a well-known anti-inflammatory interleukin, IL-10, was inserted in the construct in the place of GFP. IL-10 was chosen based on its strong anti-inflammatory properties and its ability to polarize microglial cells towards a neuroprotective phenotype, commonly known as "M2" (Orihuela et al., 2016). After verifying its expression in vitro and in vivo with qPCR and ELISA analysis, the lentiviral vector was used in vivo, returning encouraging preliminary results. In fact, neurodegeneration is reduced in animals in which the inoculation of the fibrils is followed by the expression of IL10. For an in-depth analysis of the pathway involved in the neuroprotective effect of IL10, it was necessary to study the gene expression profile through BULK RNA sequencing, thanks to which it was demonstrated that the overexpression of IL-10 by microglia has profound impact in both, microglial phenotype and in the cell survival programs of SN neurons.
Una delle sfide più complesse nella ricerca clinica è quella di sviluppare dei modelli sperimentali animali che siano in grado di ricapitolare le caratteristiche fisiopatologie della malattia umana. Per la malattia di Parkinson (MP) negli anni sono stati generati diversi modelli: da quelli tossicologici (con MPTP, 6-OHDA, rotenone) a quelli transgenici e, più recentemente, modelli basati sul comportamento simil-prionico dell’α-sinucleina (α-Syn) (Cenci & Björklund, 2020). Questi sono utilizzati non solo per comprendere i meccanismi alla base dello sviluppo e progressione della malattia, ma anche per determinare l’efficacia di un determinato approccio terapeutico. A questo proposito, questa tesi si pone l’obbiettivo di caratterizzare un nuovo modello sperimentale animale, basato sull’utilizzo di fibrille preformate (PFFs) di α-sinucleina (α-Syn) iniettate direttamente nella SN di topi C57BL. Le PPFs, una volta raggiunti i neuroni, danno luogo al processo di nucleazione dell’α-Syn endogena il quale porta alla formazione degli aggregati proteici tipici della MP. I risultati ottenuti mostrano, innanzitutto, la capacità delle PFFs utilizzate di provocare l’aggregazione dell’α-Syn endogena, sia nel sito di iniezione che nelle strutture ad esso direttamente connesse. L’aggregazione inizia già a due settimane dall’iniezione e rimane costante nel tempo. Si assiste poi a un progressivo aumento della neurodegenerazione dei neuroni dopaminergici, caratteristica fondamentale in un modello di MP. Infine, il parametro fondamentale analizzato è stata la neuroinfiammazione. Essa è stata misurata in termini di attivazione microgliale essendo questa la popolazione cellulare che interviene per prima in caso di patologie, lesioni o infezioni. Andando a misurare la presenza di marcatori microgliali quali IBA1 e CD68 è stata confermata la presenza di una forte attivazione microgliale già a partire dai time point più precoci, che va poi diminuendo nel tempo. Proprio sul ruolo della microglia si sviluppa la seconda parte di questo progetto: con l’obbiettivo di sviluppare una terapia genica è stata sfruttata la strategia del “microRNA detargeting” per ottenere l’espressione di un vettore lentivirale esclusivamente nelle cellule microgliali. Per fare questo ci si è serviti di una particolare sequenza, detta mirTAG, la quale risulta complementare a quella di specifici microRNA presenti nei diversi tipi cellulari del parenchima (neuroni, astrociti, oligodendrociti, endotelio) ma pressoché assenti nella microglia. In questo modo, il genoma lentivirale non viene degradato e può esprimersi soltanto in queste ultime. Il costrutto è stato inizialmente testato con la green fluorescent protein (GFP) sia in vitro che in vivo in modo da confermarne la specificità di espressione. Successivamente, nel costrutto è stato inserito al posto di GFP il gene di una nota interleuchina antiinfiammatoria, IL-10. Quest’ultima è stata scelta sulla base del sue spiccate proprietà antiinfiammatorie e la sua capacità di polarizzare le cellule microgliali verso un fenotipo neuroprotettivo, comunemente detto “M2” (Orihuela et al., 2016). Dopo aver verificato la sua espressione in vitro e in vivo con analisi di qPCR ed ELISA, il vettore lentivirale è stato utilizzato in vivo restituendo degli incoraggianti risultati preliminari. La neurodegenerazione risulta infatti ridotta negli animali in cui all’inoculazione delle fibrille è seguita l’espressione di IL10. Per un’approfondita analisi del pathway coinvolto nell’effetto neuroprotettivo di IL10, si è reso necessario lo studio del profilo di espressione genica tramite BULK RNA sequencing, grazie al quale è stato dimostrato come la sovraespressione di IL10 da parte della microglia abbia profonde ripercussioni sia nel modificare il fenotipo microgliale, sia nell’indurre programmi di sopravvivenza cellulare nei neuroni della SN.
Riprogrammazione del fenotipo microgliale come approccio terapeutico per ridurre la perdita di neuroni dopaminergici in un modello di aggregazione di α-sinucleina
GAMBARÈ, DIANA
2020/2021
Abstract
One of the most complex challenges in clinical research is the development of experimental animal models able to reproduce the physiopathological features of human diseases as closely as possible. Regarding Parkinson's disease (PD), several models have been generated over the years: from toxicological ones (with MPTP, 6-OHDA, rotenone) (Schober, 2004; Sherer et al., 2003) to transgenic ones (Dawson et al., 2010; Masliah et al., 2000) and, more recently, models based on the prion-like behaviour of α-synuclein (α-Syn) (Cenci & Björklund, 2020). These are used not only to understand the mechanisms underlying the development and progression of the disease, but also to determine the effectiveness of a given therapeutic approach. Therefore, this thesis aims to characterize a new animal experimental model, based on the use of preformed α-synuclein (α-Syn) fibrils (PFFs) injected directly into the SN of C57BL mice. Once they reach the neurons, the PFFs trigger the nucleation process of the endogenous α-Syn, which leads to the formation of protein aggregates typical of PD. The results obtained show, firstly, the ability of the PFFs to cause the aggregation of endogenous α-Syn, both at the injection site and in the structures directly connected to it. The aggregation is already visible two weeks after the injection and remains constant over time. There is also a progressive increase in the neurodegeneration of dopaminergic neurons, a fundamental feature in a PD model. Finally, the fundamental parameter analysed was the neuroinflammation. Given that microglia is the most important cell type in the brain involved in the resolution of damage caused by pathologies, injuries, or infections, we decided to measured the microglial activation. By measuring the presence of microglial markers such as IBA1 and CD68, the presence of a strong microglial activation was confirmed at the earliest time points, which is followed by a decrease after four months. The second part of this project focuses specifically on the role of microglia with the view to develop an effective gene therapy approach based on the "microRNA detargeting" strategy. With this in mind, we generate a lentiviral vector which allows the transgene expression exclusively in the microglial cells. To do this, a particular sequence was used, called mirTAG, which is complementary to a specific microRNAs present in different cell types (neurons, astrocytes, oligodendrocytes, endothelium) but almost absent in the microglia. In this way, the lentiviral genome is preserved only in microglia where, therefore its expression is permitted. The construct was initially tested with green fluorescent protein (GFP) both in vitro and in vivo in order to confirm the specificity of expression. Then, the gene of a well-known anti-inflammatory interleukin, IL-10, was inserted in the construct in the place of GFP. IL-10 was chosen based on its strong anti-inflammatory properties and its ability to polarize microglial cells towards a neuroprotective phenotype, commonly known as "M2" (Orihuela et al., 2016). After verifying its expression in vitro and in vivo with qPCR and ELISA analysis, the lentiviral vector was used in vivo, returning encouraging preliminary results. In fact, neurodegeneration is reduced in animals in which the inoculation of the fibrils is followed by the expression of IL10. For an in-depth analysis of the pathway involved in the neuroprotective effect of IL10, it was necessary to study the gene expression profile through BULK RNA sequencing, thanks to which it was demonstrated that the overexpression of IL-10 by microglia has profound impact in both, microglial phenotype and in the cell survival programs of SN neurons.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/13159