The objective of my thesis project was to understand mechanisms of neurodegeneration mediated by glial cells and characterize the molecular events involved in neuroprotection by Nerve Growth Factor (NGF). The interest in glial cells is based on the new concept of tripartite synapses and the existence of a bidirectional communication between neurons and astrocytes. For this study, we used a model of in-vitro reactive gliosis based on pure populations of astrocytes and/or microglia activated by Lipopolysaccharide (LPS) or the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNFα). Initially, this system was used to study the effects of glial activation on astrocytes proliferation and evaluate the anti-gliosis activity of Nerve Growth Factor (NGF). Experiments of growth rate and BrdU incorporation showed that both LPS and TNFα determined a significant increase in astrocytes proliferation, which was reduced by co-treatment with NGF. At molecular level, we studied expression levels of NGF receptors: TrkA and p75. Our data indicated that treatments with LPS increased expression levels of p75 and decreased TrkA levels. This effect was prevented by co-treatment with NGF. We also studied the effects of glial activation on neuronal survival and functions by using a model of cortical neurons exposed to conditioned medium (CM) from activated astrocytes following treatment with LPS or TNFα. In fact, treatment with the CM from activated astrocytes decreased the survival of neurons and significantly reduced the levels of TrkA, but not in the presence of NGF. It was also seen that CM from glial cells activated with TNFα, induced a significant increase of ROS, that was completely inhibited by pre-incubation (ON) with NGF. A potential mechanism of toxicity may be mediated by pro-NGF (precursor of NGF), through the increase of metalloproteinase- 9 (MMP-9), the main protease responsible for maturation and degradation of NGF. RT-PCR analysis also led to the identification of a series of genes that participate to synaptic function and are down- or up-regulated by the treatment with CM and NGF. Finally, we assessed the effect of NGF on astrocytic mitochondrial function. Preliminary experiments, by Seahorse, indicated that NGF increased the respiratory capacity of astrocytes both in basal conditions and under stress conditions. In parallel, we studied the role of the purinergic system in neuroinflammation. Indeed, several studies have shown that activation of P2X7 receptors (a class of purinergic receptors), induces the secretion of pro-inflammatory cytokines and chemokines, such as TNFα, and also stimulates the production of superoxides in microglial cells. The effect of the purinergic system on glial activation was assessed by using OxATP, a P2X7 receptor antagonist. Our data showed that chronic co-treatment with OxATP significantly prevented the proliferation of astrocytes. Furthermore, treatment with OxATP had no effect on neuronal survival and astrocytes, but prevented the production of ROS. Finally, OxATP inhibited the decrease of astrocytic and neuronal vGLUT, the glutamate transporter, both in neurons and activated astrocytes. In conclusion, these studies identify two main mechanisms of glia-mediated neurodegeneration: one based on the alteration of neurotrophic support by NGF, the other due to activation of the purinergic system.

L’obiettivo del progetto di tesi è stato quello di comprendere i meccanismi di neurodegenerazione mediati dalle cellule gliali e caratterizzare gli eventi molecolari implicati nella neuroprotezione da parte del Nerve Growth Factor (NGF). L’interesse per le cellule gliali si basa sul nuovo concetto di sinapsi tripartita che afferma l’esistenza di una comunicazione bidirezionale tra neuroni e astrociti. Per questo studio, è stato utilizzato un modello di gliosi reattiva in-vitro basato su popolazioni pure di astrociti e/o microglia attivati mediante trattamento con Lipolissacaride (LPS) o con la citochina pro-infiammatoria TNF-α. Inizialmente, questo sistema è stato usato per studiare gli effetti dell’attivazione gliale sulla proliferazione e l’attività anti-gliosi del NGF, come da precedenti studi in-vivo. Esperimenti di curva di crescita e di incorporazione di BrdU mostrano che sia LPS che TNFα determinano un significativo aumento della proliferazione astrocitaria, che viene ridotto dal co-trattamento con NGF. Per capire il meccanismo mediante il quale il NGF agisce, sono stati studiati i livelli di espressione dei recettori: TrkA e p75. In effetti, i dati ottenuti indicano che i trattamenti con LPS aumentano i livelli di espressione di p75 e diminuiscono i livelli di TrkA. Questo effetto è inibito dal cotrattamento con NGF. Contemporaneamente è stato studiato l’effetto dell’attivazione gliale su sopravvivenza e funzionalità neuronale utilizzando un modello di neuroni corticali esposti a medium condizionato (CM) da astrociti attivati in seguito a trattamento con LPS o TNFα. In effetti, il trattamento con il CM da astrociti attivati diminuisce la sopravvivenza dei neuroni e riduce significativamente i livelli di TrkA, ma non in presenza di NGF. Inoltre, Il NGF inibisce l’aumento di ROS indotto dal trattamento con CM da cellule gliali attivate con TNFα. Un potenziale meccanismo di tossicità potrebbe essere mediato dall’aumento di pro-NGF (precursore del NGF) e metalloproteinasi-9 (MMP-9), responsabile della maturazione e della degradazione del NGF. L’analisi RT-PCR, inoltre, ha portato all’identificazione di diversi geni associati alla funzionalità sinaptica e che sono down- o up-regolati dal trattamento con CM o con NGF. Infine, è stato valutato l’effetto del NGF sulla funzionalità mitocondriale degli astrociti. Esperimenti preliminari, effettuati tramite Seahorse, indicano che il NGF aumenta la capacità respiratoria degli astrociti sia in condizioni basali che in condizioni di stress. Contemporaneamente sono stati svolti alcuni studi sul ruolo del sistema purinergico nella neuroinfiammazione. L’effetto del sistema purinergico sull’attivazione degli astrociti è stato valutato tramite OxATP, antagonista dei recettori P2X7. I dati mostrano che il co-trattamento cronico con OxATP previene in maniera significativa la proliferazione degli astrociti. Inoltre, il trattamento con OxATP non ha effetto sulla sopravvivenza neuronale e astrocitaria, ma previene la produzione di ROS. Infine, OxATP inibisce la riduzione dei livelli di vGLUT, il trasportatore del glutammato, sia nei neuroni che negli astrociti attivati. Nel complesso, questi studi identificano due meccanismi di neurodegenerazione mediata dalla glia: uno legato ad alterazione del sistema di supporto neurotrofico da parte del NGF, l’altro legato ad eccessiva attivazione del sistema purinergico.

(2015). Mechanisms of glia-mediated neurodegeneration: modulation by Nerve Growth Factor (NGF) and the purinergic system. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).

Mechanisms of glia-mediated neurodegeneration: modulation by Nerve Growth Factor (NGF) and the purinergic system

IPPOLITO, VITA MARIA
2015

Abstract

The objective of my thesis project was to understand mechanisms of neurodegeneration mediated by glial cells and characterize the molecular events involved in neuroprotection by Nerve Growth Factor (NGF). The interest in glial cells is based on the new concept of tripartite synapses and the existence of a bidirectional communication between neurons and astrocytes. For this study, we used a model of in-vitro reactive gliosis based on pure populations of astrocytes and/or microglia activated by Lipopolysaccharide (LPS) or the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNFα). Initially, this system was used to study the effects of glial activation on astrocytes proliferation and evaluate the anti-gliosis activity of Nerve Growth Factor (NGF). Experiments of growth rate and BrdU incorporation showed that both LPS and TNFα determined a significant increase in astrocytes proliferation, which was reduced by co-treatment with NGF. At molecular level, we studied expression levels of NGF receptors: TrkA and p75. Our data indicated that treatments with LPS increased expression levels of p75 and decreased TrkA levels. This effect was prevented by co-treatment with NGF. We also studied the effects of glial activation on neuronal survival and functions by using a model of cortical neurons exposed to conditioned medium (CM) from activated astrocytes following treatment with LPS or TNFα. In fact, treatment with the CM from activated astrocytes decreased the survival of neurons and significantly reduced the levels of TrkA, but not in the presence of NGF. It was also seen that CM from glial cells activated with TNFα, induced a significant increase of ROS, that was completely inhibited by pre-incubation (ON) with NGF. A potential mechanism of toxicity may be mediated by pro-NGF (precursor of NGF), through the increase of metalloproteinase- 9 (MMP-9), the main protease responsible for maturation and degradation of NGF. RT-PCR analysis also led to the identification of a series of genes that participate to synaptic function and are down- or up-regulated by the treatment with CM and NGF. Finally, we assessed the effect of NGF on astrocytic mitochondrial function. Preliminary experiments, by Seahorse, indicated that NGF increased the respiratory capacity of astrocytes both in basal conditions and under stress conditions. In parallel, we studied the role of the purinergic system in neuroinflammation. Indeed, several studies have shown that activation of P2X7 receptors (a class of purinergic receptors), induces the secretion of pro-inflammatory cytokines and chemokines, such as TNFα, and also stimulates the production of superoxides in microglial cells. The effect of the purinergic system on glial activation was assessed by using OxATP, a P2X7 receptor antagonist. Our data showed that chronic co-treatment with OxATP significantly prevented the proliferation of astrocytes. Furthermore, treatment with OxATP had no effect on neuronal survival and astrocytes, but prevented the production of ROS. Finally, OxATP inhibited the decrease of astrocytic and neuronal vGLUT, the glutamate transporter, both in neurons and activated astrocytes. In conclusion, these studies identify two main mechanisms of glia-mediated neurodegeneration: one based on the alteration of neurotrophic support by NGF, the other due to activation of the purinergic system.
COLANGELO, ANNA MARIA
Glia, Reactive gliosis, NGF, P2X7 Receptors
BIO/10 - BIOCHIMICA
English
13-feb-2015
Scuola di dottorato di Scienze
BIOTECNOLOGIE INDUSTRIALI - 15R
27
2013/2014
open
(2015). Mechanisms of glia-mediated neurodegeneration: modulation by Nerve Growth Factor (NGF) and the purinergic system. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).
File in questo prodotto:
File Dimensione Formato  
PhD_unimib_760903.pdf

accesso aperto

Descrizione: Tesi di dottorato
Tipologia di allegato: Doctoral thesis
Dimensione 1.83 MB
Formato Adobe PDF
1.83 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/70478
Citazioni
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
Social impact