| dc.description.abstracteng | Several neurodegenerative disorders, such as Parkinson’s disease (PD), are characterized by the deposition of misfolded and aggregated forms of a particular protein in different areas of the brain. Understanding the molecular mechanisms of neurodegenerative diseases are extremely important to prevent and stop such debilitate diseases.
PD is a movement related disorder that primarily affects aged individuals, but mutations on alpha-Synuclein (aSyn) gene (SNCA) have been identified in an early- and juvenile-onset of the disease.
aSyn is a small an intrinsically disorder protein, that binds to membrane and lipids. It is the major component of Lewy Bodies (LBs) and Lewy Neurites (LN) in the surviving neurons in parkinsonian brains. However, the molecular mechanisms that lead to the selective degeneration of dopaminergic neurons from the substantia nigra pars compacta are still unclear. aSyn aggregation is an important process for the pathology. In pathological conditions, aSyn aggregates, forming oligomeric species that can rapidly convert into amyloid fibrils. Amyloid fibrils made up of aSyn then deposit in LBs, along with several other proteins and lipids. A combination of in vitro, cell and animal models studies has been useful to investigate not only aSyn aggregation intermediates, but also the toxic mechanisms.
Over the years, the number of models in the PD field increased significantly, but has not generated consensus with respect to the best models to use. Thus, it is important to choose the appropriate models to investigate a particular question of interest. Roughly, the PD models can be divided into two categories: those based on genetic alterations and those based on the effect of toxins. For the purpose of this thesis, I will focus on the genetic models. Due to our limited understanding of the molecular mechanisms underlying disease, cell-based models constitute a powerful tool to mimic important aspects of basic aSyn biology (such as aggregation and toxicity), and afford unique opportunities to test the effect of possible therapeutic strategies. A number of molecular dysfunctions have been associated with PD. These include defects in mitochondrial function, defects in degradation systems (ubiquitin–proteasome and autophagy), increased production of reactive oxygen species, or impairment of intracellular trafficking. However, the lack of systematic comparisons
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makes it difficult to interpret and reconcile the relevance of results obtained using different models.
This study focuses mainly on the exploitation of two cell-based systems that try to model the oligomerization and aggregation of aSyn. The Bimolecular Fluorescence complementation assay enables us to visualize the dimerization and oligomerization of aSyn in living cells. To model aSyn aggregation, manipulations of the C-terminal region have been useful, as they promote the formation of LBs-like inclusions that can be readily detected by immunostaining. Thus, using these two models, we performed a systematic comparison to investigate the effects of genetic alterations on aSyn oligomerization and aggregation, and how these impacted on selected cellular functions. In addition, we investigated the effects of small molecules on the conformation and aggregation of aSyn, as this is thought to be a possible target for therapeutic intervention.
Overall, our studies demonstrated the usefulness of the two cell-based models for studying specific aspects of aSyn aggregation and for the screening of drugs that may lead to the identification of novel therapeutic strategies for PD and other synucleinopathies. | de |