title: Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells.
abstract: BACKGROUND
  Alpha-synuclein oligomerization is associated to Parkinson's disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease.
  
  
  METHODS
  We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging.
  
  
  RESULTS
  Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria may be dysfunctional.
  
  
  CONCLUSIONS
  We showed that alpha-synuclein overexpression in SH-SY5Y causes the formation of alpha-synuclein oligomeric species, whose presence is associated with mitochondrial fragmentation and autophagic-lysosomal pathway activation in live cells.
  
  
  CONCLUSIONS
  The unique capability provided by the Number and Brightness analysis to study alpha-synuclein oligomer distribution and properties, and the study of their association to intracellular components in single live cells is important to forward our understanding of the molecular mechanisms of Parkinson's disease and it may be of general significance when applied to the study of other aggregating proteins in cellular models.
authors.affiliation: Department of Biology, University of Padova, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Plotegher
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, USA.
authors.forename: E
authors.initials: E
authors.lastname: Gratton
authors.affiliation: Department of Biology, University of Padova, Italy. Electronic address: luigi.bubacco@unipd.it.
authors.forename: L
authors.initials: L
authors.lastname: Bubacco
mesh_terms: D002138:Calibration; D045744:Cell Line, Tumor; D002470:Cell Survival; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008928:Mitochondria / Q000473:pathology*; D055503:Protein Multimerization; D051844:alpha-Synuclein / Q000737:chemistry*
chemical_list: D051844:alpha-Synuclein
keywords: Fluorescence; Live imaging; Lysosomes; Oligomers; Parkinson's disease; Protein aggregation