Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

Summary

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

Abstract

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer's disease (AD), Parkinson's disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of β-amyloid (Aβ), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

Introduction

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and the closely related prion diseases are proteinopathies characterized by the gradual accumulation of detergent-insoluble protein aggregates in the brain with accompanying cognitive decline.^1,2 This shared pathological feature is thought to play a central role in the etiology and pathophysiology of these neurodegenerative diseases.² These aggregates typically consist of polymeric amyloid fibers, which are composed of repeati....

Protocol

Ethics Statement: All brain tissues were obtained from the Emory Alzheimer's Disease Research Center (ADRC) Brain Bank. Human postmortem tissues were acquired under proper Institutional Review Board (IRB) protocols.

1. Homogenization and Fractionation

1. Tissue selection
   NOTE: Frozen postmortem frontal cortex tissue from healthy control (Ctl) and pathologically confirmed AD cases were selected from the Emory ADRC brain bank (n=2). Post-mortem neuropathological evaluation of amyloid plaque distribution was performed according to the Consortium to Establish a Registry for Alzheimer's Disease s....

Results

The abbreviated single-step sarkosyl-fractionation protocol was used to enrich detergent-insoluble protein aggregates from control and AD postmortem brain (Figure 1). Proteins from TH-S, S1, S2 and P2 fractions were resolved by SDS-PAGE, fixed for 15 min in Coomassie blue fixative buffer followed by gentle staining with Coomassie Brilliant Blue G-250 staining buffer. The resuspension step is optional since there were undetectable levels of protein in the S2 f.......

Discussion

Herein we introduce and discuss an abbreviated single-step detergent-fractionation protocol that is applicable to a wide variety of experimental applications ranging from mass spectrometry-based proteomics analysis to functional protein misfolding assays and western blotting.^5,6,7,10 This methodology is perhaps most effective when used to study neurodegenerative proteinopathies such as Alzheime.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Protease and phosphatase inhibitor cocktail, EDTA-free (100X)	Thermo Fisher	78441	protease & phosphatase inhibitor cocktail
Sonic Dismembrator System (ultrasonicator)	Fisher Scientific	FB505110	microtip ultrasonicator
Optimax TLX Ultracentrifuge	Beckman Coulter	361545	refrigerated ultracentrifuge
TLA120.1 rotor	Beckman Coulter	362224	ultracentrifuge rotor
500 ul (8 x 34 mm) polycarbonate tubes, thickwall	Beckman Coulter	343776	ultracentrifuge tubes for TLA120.1 rotor
4X SDS sample buffer	Home-made	N/A	SDS-PAGE
TCEP solution, neutral pH	Thermo Fisher	77720	reducing agent
(TBS) blocking buffer	Thermo Fisher	37542	blocking buffer
(TBS) blocking buffer + 0.05% Tween 20	Thermo Fisher	37543	blocking buffer and antibody diluent
4-12% Bolt Bis-Tris Plus gels, 10-well	Thermo Fisher	NW04120BOX	precast SDS-PAGE gels
MES SDS Running Buffer (20X)	Thermo Fisher	B0002	SDS-PAGE running buffer
N-Lauroylsarcosine sodium salt (sarkosyl)	Sigma Aldrich	L5777-50G	detergent
Anti-Tau-2 (pan tau) antibody	Chemicon	MAB375	antibodies
Anti-phospho-threonine 231 Tau antibody	Millipore	MAB5450	antibodies
Anti-phospho-seroine 202 and threonine 205 Tau antibody (AT8)	Thermo Fisher	MN1020	antibodies
Anti-early endosome antigen 1 (EEA1) antibody	abcam	ab2900	antibodies
Alexa Fluor 680 goat anti-mouse IgG (H+L) secondary antibody	Thermo Fisher	A21058	antibodies
Alexa Fluor 790 donkey anti-rabbit IgG (H+L) secondary antibody	Thermo Fisher	A11374	antibodies
iBlot2 Dry Blotting System	Thermo Fisher	IB21001	Gel transfer
iBlot2 Transfer Stacks, Nitrocellulose, mini	Thermo Fisher	IB23002	Gel transfer