TY - JOUR
T1 - Purification of α-synuclein containing inclusions from human post mortem brain tissue
AU - Mccormack, Amellia
AU - Chegeni Farahani, Nusha
AU - Chegini, Fariba
AU - Colella, Alexander
AU - Power, John
AU - Keating, Damien
AU - Chataway, Timothy
PY - 2016/6/15
Y1 - 2016/6/15
N2 - Comparison with existing methods. Background: Neurodegenerative disorders affect a large proportion of the elderly population. A group of disorders, known as the α-synucleinopathies, are characterised by the presence of α-synuclein-containing protein inclusions, such as Lewy Bodies (LBs) found in neurons from Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB), and Glial Cytoplasmic Inclusions (GCIs) found in oligodendrocytes from Multiple System Atrophy (MSA). The analysis of the protein composition of inclusions has been hindered by limitations of methods for isolating the inclusions from the surrounding tissue. Method: Four modifications were made to the published method for GCI purification by Gai et al. (1999) which were: collecting the entire inclusion-containing part of the Percoll gradient; lysis of nuclei prior to DNAse digestion; limited tryptic digestion to release inclusions from the cytoskeletal meshwork; and increased antibody and magnetic bead concentrations/volumes to capture the larger amounts of inclusions. Results: The optimised method gave a 28-fold increase in yield compared to the published method of Gai et al. (1999). A 2D-DIGE comparison revealed a 3.8-fold increase in α-synuclein enrichment and a corresponding 5.2-fold reduction in tubulin contamination. This method was also successfully adapted to the purification of LBs from DLB tissue.A 2D-DIGE comparison of purified GCIs (n = 2) revealed that GCIs consist of 11.7% α-synuclein, 1.9% α-β-crystallin and 2.3% 14-3-3 proteins compared to 8.5%, 2.0% and 1.5% in LBs, respectively. Conclusions: This study has generated an improved method for the purification of α-synuclein-containing inclusions with a yield sufficient for multiple forms of analysis.
AB - Comparison with existing methods. Background: Neurodegenerative disorders affect a large proportion of the elderly population. A group of disorders, known as the α-synucleinopathies, are characterised by the presence of α-synuclein-containing protein inclusions, such as Lewy Bodies (LBs) found in neurons from Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB), and Glial Cytoplasmic Inclusions (GCIs) found in oligodendrocytes from Multiple System Atrophy (MSA). The analysis of the protein composition of inclusions has been hindered by limitations of methods for isolating the inclusions from the surrounding tissue. Method: Four modifications were made to the published method for GCI purification by Gai et al. (1999) which were: collecting the entire inclusion-containing part of the Percoll gradient; lysis of nuclei prior to DNAse digestion; limited tryptic digestion to release inclusions from the cytoskeletal meshwork; and increased antibody and magnetic bead concentrations/volumes to capture the larger amounts of inclusions. Results: The optimised method gave a 28-fold increase in yield compared to the published method of Gai et al. (1999). A 2D-DIGE comparison revealed a 3.8-fold increase in α-synuclein enrichment and a corresponding 5.2-fold reduction in tubulin contamination. This method was also successfully adapted to the purification of LBs from DLB tissue.A 2D-DIGE comparison of purified GCIs (n = 2) revealed that GCIs consist of 11.7% α-synuclein, 1.9% α-β-crystallin and 2.3% 14-3-3 proteins compared to 8.5%, 2.0% and 1.5% in LBs, respectively. Conclusions: This study has generated an improved method for the purification of α-synuclein-containing inclusions with a yield sufficient for multiple forms of analysis.
KW - Dementia with lewy bodies
KW - Glial cytoplasmic inclusion
KW - Lewy body
KW - Multiple system atrophy
KW - Parkinson's disease
KW - Purification
UR - http://www.scopus.com/inward/record.url?scp=84962910565&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2016.03.016
DO - 10.1016/j.jneumeth.2016.03.016
M3 - Article
VL - 266
SP - 141
EP - 150
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
ER -