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

Abstract

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 proteinmore » 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.« less

Authors:
 [1];  [2];  [3];  [1];  [4];  [3]
  1. Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany)
  2. (Germany)
  3. Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany)
  4. Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224 (United States)
Publication Date:
OSTI Identifier:
22606147
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 477; Journal Issue: 1; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; APOPTOSIS; FLUORESCENCE; HEAT; HEAT-SHOCK PROTEINS; IN VITRO; INHIBITION; MOLECULES; NERVOUS SYSTEM DISEASES; PATHOGENESIS; SENSITIVITY; TOXICITY

Citation Formats

Levin, Johannes, German Center for Neurodegenerative Diseases – DZNE, Site Munich, Feodor-Lynen-Str. 17, 81377 Munich, Hillmer, Andreas S., Högen, Tobias, McLean, Pamela J., and Giese, Armin, E-mail: armin.giese@med.uni-muenchen.de. Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.06.023.
Levin, Johannes, German Center for Neurodegenerative Diseases – DZNE, Site Munich, Feodor-Lynen-Str. 17, 81377 Munich, Hillmer, Andreas S., Högen, Tobias, McLean, Pamela J., & Giese, Armin, E-mail: armin.giese@med.uni-muenchen.de. Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy. United States. doi:10.1016/J.BBRC.2016.06.023.
Levin, Johannes, German Center for Neurodegenerative Diseases – DZNE, Site Munich, Feodor-Lynen-Str. 17, 81377 Munich, Hillmer, Andreas S., Högen, Tobias, McLean, Pamela J., and Giese, Armin, E-mail: armin.giese@med.uni-muenchen.de. 2016. "Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy". United States. doi:10.1016/J.BBRC.2016.06.023.
@article{osti_22606147,
title = {Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy},
author = {Levin, Johannes and German Center for Neurodegenerative Diseases – DZNE, Site Munich, Feodor-Lynen-Str. 17, 81377 Munich and Hillmer, Andreas S. and Högen, Tobias and McLean, Pamela J. and Giese, Armin, E-mail: armin.giese@med.uni-muenchen.de},
abstractNote = {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.},
doi = {10.1016/J.BBRC.2016.06.023},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 477,
place = {United States},
year = 2016,
month = 8
}
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  • Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). {alpha}-Synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and {alpha}B-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messengermore » RNA levels are {approx}2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by {approx}80% in a culture model while {alpha}B-crystallin reduces toxicity by {approx}20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model.« less