Purification of α-synuclein containing inclusions from human post mortem brain tissue

Area of Science:

• Neuroscience
• Biochemistry
• Pathology

Background:

• Neurodegenerative disorders, including Parkinson's Disease (PD) and Multiple System Atrophy (MSA), are characterized by α-synuclein protein inclusions.
• Analyzing the protein composition of these inclusions (Lewy Bodies and Glial Cytoplasmic Inclusions) has been challenging due to limitations in isolation methods.

Purpose of the Study:

• To develop an optimized method for purifying α-synuclein-containing inclusions.
• To improve the yield and purity of isolated inclusions for subsequent proteomic analysis.

Main Methods:

• Modified the published GCI purification method by Gai et al. (1999).
• Key modifications included optimizing gradient collection, nuclear lysis, enzymatic digestion, and antibody/bead concentrations.
• Adapted the method for purifying Lewy Bodies (LBs) from Dementia with Lewy Bodies (DLB) tissue.

Main Results:

• Achieved a 28-fold increase in yield compared to the original method.
• Demonstrated a 3.8-fold increase in α-synuclein enrichment and a 5.2-fold reduction in tubulin contamination.
• Identified distinct protein compositions in Glial Cytoplasmic Inclusions (GCIs) and Lewy Bodies (LBs) using 2D-DIGE.

Conclusions:

• An improved method for purifying α-synuclein inclusions has been established.
• The enhanced yield and purity facilitate comprehensive proteomic analysis of inclusions.
• This method is applicable to inclusions from various α-synucleinopathies, including PD, DLB, and MSA.