CHARACTERIZATION OF α-SYNUCLEIN AGGREGATION, NEURODEGENERATION AND NEUROINFLAMMATION IN MODELS OF PARKINSON’S DISEASE

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by bradykinesia, resting tremor and muscle rigidity. The pathological hallmarks of PD are the loss of the dopaminergic (DA) neurons in the substantia nigra (SN) and the presence of intracellular protein inclusions called Lewy bodies (LBs).

Alpha-synuclein (α-SYN) is a small protein of 140 amino acids, which is ubiquitously expressed in the brain. α-SYN is considered a key player in the pathogenesis of PD: first, aggregated α-SYN was shown to be the major component of LBs; second, point mutations and multiplications of α-SYN gene were identified as cause of autosomal dominant forms of PD. Moreover, there is evidence that under pathological conditions α-SYN is able to aggregate into soluble oligomers, protofibrils and finally insoluble β-sheet rich fibrils, however the exact relationship between α-SYN aggregation and neurodegeneration remains under debate. Therefore, the availability of cellular assays that allow medium-throughput analysis of α-SYN-linked pathology would be extremely useful to study the aggregation process and to develop α-SYN-based therapies. For this reason, in the first part of the thesis we described the optimization of a high-content neuronal cell assay that simultaneously measures oxidative stress-induced α-SYN aggregation and apoptosis in human SH-SY5Y neuroblastoma cells. The quantification of α-SYN aggregates in cells has commonly relied on manual imaging and counting, which are time consuming and do not allow a concurrent analysis of cell viability. Our high-content analysis (HCA) method for quantification of α-SYN aggregation allows simultaneous measurements of multiple cell parameters at a single-cell level in a fast and automated manner, therefore offering an objective and multiparametric approach for the screening of compounds and genes that might alter α-SYN aggregation and toxicity.

Although the mechanisms contributing to dopaminergic (DA) cell death in PD are not completely elucidated, several evidences support the involvement of neuroinflammation in α-SYN induced neurodegeneration.  Microglial cells constitute the innate immune system of the brain. They exist in a resting state with a ramified morphology and monitor the environment for signs of distress. Activated microglia can adopt two different phenotypes with distinct functions. Classically activated microglial cells produce damaging reactive oxygen species (ROS) and secrete pro-inflammatory cytokines that bind to neuronal receptors and initiate cell death pathways, while alternatively activated microglia display a repair function by upregulating the production of anti-inflammatory cytokines and the release of trophic factors. Evidence for microglial activation in SN of PD patients arise from post mortem and PET imaging studies. Moreover, several α-SYN cellular assays and PD animal models show microglial activation and proliferation before the onset of DA neurodegeneration, suggesting an active contribution of the innate immune response to PD progression. However, the α-SYN assemblies responsible for the induction and maintenance of microglial activation in PD patients and animal models have not been identified yet. The most widely accepted hypothesis is that α-SYN oligomers represent the neurotoxic assemblies in PD, however recent evidence showing spreading of fibrillar α-SYN from one cell to the other in brains of PD patients, in cell culture and in PD animal models, supports a role for α-SYN fibrils in PD pathogenesis. Therefore, in order to define the α-SYN assemblies mediating the neuroinflammatory response, we analysed the activation profile of microglial cells upon exogenous administration of α-SYN monomers, oligomers and two structurally different types of α-SYN fibrils. We showed that only the fibrillar α-SYN assemblies induce the activation of microglia and the release of pro-inflammatory cytokines both in a microglial cell line and in murine primary microglia. Moreover, we showed that the injection of fibrillar α-SYN in mouse SN induces a strong microglial response, while oligomeric α-SYN only gives a mild microglial recruitment. These data suggest that the activation of the innate immune system by α-SYN fibrils might be a possible mechanism contributing to neuronal cell death.