Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy

Journal Article · Fri Aug 12 04:00:00 EDT 2016 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2016.06.023· OSTI ID:22606147

Levin, Johannes ^[1]; Hillmer, Andreas S. ^[2]; Högen, Tobias ^[1]; McLean, Pamela J. ^[3]; Giese, Armin ^[2]

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1. Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany)
2. Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany)
3. Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224 (United States)

Synucleinopathies such as dementia with Lewy bodies or Parkinson’s disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation. - Highlights: • Single particle spectroscopy detects intracellular formed α-synuclein aggregates. • Fusion proteins allow detection of protein aggregates at the oligomer level. • The technique detects molecules inhibiting α-synuclein aggregate formation. • Single particle spectroscopy is suited for high throughput measurements.

• OSTI ID: 22606147
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 1 Vol. 477; ISSN 0006-291X; ISSN BBRCA9
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
APOPTOSIS
FLUORESCENCE
HEAT
HEAT-SHOCK PROTEINS
IN VITRO
INHIBITION
MOLECULES
NERVOUS SYSTEM DISEASES
PATHOGENESIS
SENSITIVITY
TOXICITY