Misfolding and aggregation of the synaptic protein alpha-synuclein (αsyn) is, at present, considered one of the main drivers of the pathogenesis of Parkinson’s disease (PD). PD is a complex neurodegenerative disorder characterized by progressive loss of DAergic neurons in the substantia nigra pars compacta (SNpc) and deposition of insoluble proteinaceous inclusions containing αsyn, called Lewy Bodies (LB), in different regions of the nervous system. Toxic species of αsyn were demonstrated to affect multiple cellular pathways, mediating synaptic dysfunction even before a dramatic loss of SNpc neurons has occurred. Besides its neurotoxicity towards the DAergic system, αsyn has been recently reported to affect also the corticostriatal glutamatergic signalling, modulating the synaptic levels and activity of NMDA-type glutamate receptors. However, the precise molecular events underlying αsyn synaptic toxicity are still elusive. The synaptic retention of NMDARs was recently demonstrated to be strictly correlated to the interaction with the protein Rabphilin-3A (Rph3A). Interestingly, alterations of Rph3A expression and its interaction with NMDARs at the corticostriatal synapse have been described in advanced PD stages. Besides, a direct αsyn/Rph3A interaction, modulated in presence of LB, has been put forward. Starting from these previous findings, this PhD project is aimed at characterizing Rph3A role in the early stages of PD to identify pharmacological approaches able to slow down αsyn-induced synaptic toxicity. Exploiting different imaging and biochemical approaches, Rph3A was confirmed as a novel αsyn interactor at synaptic sites. In addition, using an in vivo mouse model of αsyn-induced PD, we found that oligomers and fibrils (PFF) of αsyn reduced AMPAR and NMDAR subunits at the striatal excitatory synapse, prior to cause significant DAergic neurodegeneration. At the same time point, αsyn-mice showed decreased striatal dendritic spine density and early motor impairments. Interestingly, I also found that αsyn oligomers and PFF selectively reduced striatal Rph3A postsynaptic levels and Rph3A binding to GluN2A subunit of NMDAR, suggesting Rph3A and Rph3A/αsyn complex as possible mediators of the synaptic dysfunction. Based on these hypothesis, modulatory strategies aimed at restoring Rph3A synaptic levels were firstly tested in an in vitro model of PFF-induced synaptopathy. In particular, a Rph3A/αsyn uncoupling compound (Compound B), identified through a bioinformatic screening, revealed efficacious in blocking spine loss caused by PFF exposure. In the same experimental model, synaptic defects were also prevented by Rph3A overexpression. Based on these in vitro results, these Rph3A-modulatory approaches were then investigated using the αsyn-induced PD mouse model. Notably, chronic intracerebroventricular treatment with Compound B confirmed its efficacy in blocking αsyn-dependent decrease in striatal spine density. Furthermore, striatal delivery of an AAV overexpressing Rph3A resulted sufficient in preventing the early motor impairments found in αsyn-lesioned mice. In conclusion, the results of this PhD thesis demonstrate that Rph3A and Rph3A/αsyn complex significantly contribute to the molecular events underlying early dysfunction of the striatal glutamatergic synapse in PD, therefore representing novel and promising pharmacological targets.

IDENTIFICATION OF PHARMACOLOGICAL APPROACHES BLOCKING ALPHA-SYNUCLEIN SYNAPTIC TOXICITY / E. Ferrari ; tutor: F. Gardoni ; co-tutor: I. Eberini; coordinatore: G. D. Norata. - : . Dipartimento di Scienze Farmacologiche e Biomolecolari, 2021 Dec 09. ((34. ciclo, Anno Accademico 2021.

IDENTIFICATION OF PHARMACOLOGICAL APPROACHES BLOCKING ALPHA-SYNUCLEIN SYNAPTIC TOXICITY

E. Ferrari
2021

Abstract

Misfolding and aggregation of the synaptic protein alpha-synuclein (αsyn) is, at present, considered one of the main drivers of the pathogenesis of Parkinson’s disease (PD). PD is a complex neurodegenerative disorder characterized by progressive loss of DAergic neurons in the substantia nigra pars compacta (SNpc) and deposition of insoluble proteinaceous inclusions containing αsyn, called Lewy Bodies (LB), in different regions of the nervous system. Toxic species of αsyn were demonstrated to affect multiple cellular pathways, mediating synaptic dysfunction even before a dramatic loss of SNpc neurons has occurred. Besides its neurotoxicity towards the DAergic system, αsyn has been recently reported to affect also the corticostriatal glutamatergic signalling, modulating the synaptic levels and activity of NMDA-type glutamate receptors. However, the precise molecular events underlying αsyn synaptic toxicity are still elusive. The synaptic retention of NMDARs was recently demonstrated to be strictly correlated to the interaction with the protein Rabphilin-3A (Rph3A). Interestingly, alterations of Rph3A expression and its interaction with NMDARs at the corticostriatal synapse have been described in advanced PD stages. Besides, a direct αsyn/Rph3A interaction, modulated in presence of LB, has been put forward. Starting from these previous findings, this PhD project is aimed at characterizing Rph3A role in the early stages of PD to identify pharmacological approaches able to slow down αsyn-induced synaptic toxicity. Exploiting different imaging and biochemical approaches, Rph3A was confirmed as a novel αsyn interactor at synaptic sites. In addition, using an in vivo mouse model of αsyn-induced PD, we found that oligomers and fibrils (PFF) of αsyn reduced AMPAR and NMDAR subunits at the striatal excitatory synapse, prior to cause significant DAergic neurodegeneration. At the same time point, αsyn-mice showed decreased striatal dendritic spine density and early motor impairments. Interestingly, I also found that αsyn oligomers and PFF selectively reduced striatal Rph3A postsynaptic levels and Rph3A binding to GluN2A subunit of NMDAR, suggesting Rph3A and Rph3A/αsyn complex as possible mediators of the synaptic dysfunction. Based on these hypothesis, modulatory strategies aimed at restoring Rph3A synaptic levels were firstly tested in an in vitro model of PFF-induced synaptopathy. In particular, a Rph3A/αsyn uncoupling compound (Compound B), identified through a bioinformatic screening, revealed efficacious in blocking spine loss caused by PFF exposure. In the same experimental model, synaptic defects were also prevented by Rph3A overexpression. Based on these in vitro results, these Rph3A-modulatory approaches were then investigated using the αsyn-induced PD mouse model. Notably, chronic intracerebroventricular treatment with Compound B confirmed its efficacy in blocking αsyn-dependent decrease in striatal spine density. Furthermore, striatal delivery of an AAV overexpressing Rph3A resulted sufficient in preventing the early motor impairments found in αsyn-lesioned mice. In conclusion, the results of this PhD thesis demonstrate that Rph3A and Rph3A/αsyn complex significantly contribute to the molecular events underlying early dysfunction of the striatal glutamatergic synapse in PD, therefore representing novel and promising pharmacological targets.
GARDONI, FABRIZIO
NORATA, GIUSEPPE DANILO
Parkinson's disease; alpha-synuclein; synaptopathy; neurodegeneration
Settore BIO/14 - Farmacologia
IDENTIFICATION OF PHARMACOLOGICAL APPROACHES BLOCKING ALPHA-SYNUCLEIN SYNAPTIC TOXICITY / E. Ferrari ; tutor: F. Gardoni ; co-tutor: I. Eberini; coordinatore: G. D. Norata. - : . Dipartimento di Scienze Farmacologiche e Biomolecolari, 2021 Dec 09. ((34. ciclo, Anno Accademico 2021.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/885133
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