Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Summary

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Abstract

Deposition of amyloid beta protein (Aβ) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer's disease (AD). Therefore, detection of the presence of Aβ in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and Aβ-specific antibodies have been used to detect Aβ histochemically in AD brain tissue. Use of these compounds for Aβ detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for Aβ, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of Aβ plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label Aβ plaques from both a genetic mouse model of 5x familial Alzheimer's disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as Aβ-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image Aβ plaques when compared to these conventional dyes and is comparable to Aβ-specific antibodies. In addition, Cur binds with most Aβ species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for Aβ plaques.

Introduction

Alzheimer's disease (AD) is one of the most common, age-related, progressive neurological disorders and one of the leading causes of death worldwide^1,2. Learning, memory, and cognition impairment, along with neuropsychiatric disorders, are the common symptoms manifested in AD³. Although the etiology of AD has not been fully elucidated, the available genetic, biochemical, and experimental evidence indicates that gradual deposition of Aβ is a definitive biomarker for AD⁴. This misfolded protein accumulates in intracellular and extracellular spaces and is t....

Protocol

All methods described here have been approved by the Animal Care and Use Committee (ACUC) of Saginaw Valley State University. The human tissue was obtained from an established brain bank at the Banner Sun Health Institute in Arizona^15,16.

1. Perfusion of the animals

1. Prepare the fixative and perfusion buffers.
   1. Prepare 0.1 M sodium phosphate buffer by adding 80 g of sodium chloride (NaCl), 2 g of potassium chloride (KCl), 21.7 g of disodium hydrogen phosphate (Na₂HPO₄·7H₂O), 2.59 g of potassium dihydrogen phosphate (KH_....

Results

Curcumin labels Aβ plaques within a minute. When we stained 5xFAD tissue with Cur, we found that Cur label Aβ plaques within 1 min. Although increased incubation time with Cur slightly increased the fluorescence intensity of Aβ plaques, the number of observed Aβ plaques was not significantly different between 1 min and 5 min staining time (Figure 1).

Cur can label Aβ plaques in cryostat-prepared, pa.......

Discussion

Our hypothesis was that Cur could be used as the quickest, easiest, and least expensive way to label and image Aβ plaques in postmortem AD brain tissue when compared to other classical amyloid binding dyes, as well as Aβ-specific antibodies. The aims of this study were to determine the minimum time required to label and image Aβ plaques by Cur in postmortem AD brain tissue and determine whether Cur can be used as an alternative to Aβ antibody for labeling Aβ plaques. To this end, the Aβ-labe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4′,6-diamidino-2-phenylindole (DAPI)	IHC world, Woodstock, MD
Aanimal model of Alzheimer's disease	Jackson's laboratory, Bar Harbor, ME
Absolute alcohol	VWR,Radnor, PA
Alexa 594	Santacruz Biotech, Dallas, TX
Antibody 6E10	Biolegend, San Diego, CA
Antibody A11	Millipore, Burlington, MA
Compound light microscope	Olympus, Shinjuku, Japan	Olympus BX51
Congo red	Sigma, St. Louis, MO
Cryostat	GMI, Ramsey, MN	LeicaCM1800
Curcumin	Sigma, St. Louis, MO
Disodium hydrogen phosphate	Sigma, St. Louis, MO
Dystyrene plasticizer xylene	BDH, Dawsonville, GA
Filter papers	Fisher scientific, Pittsburgh, PA
Hoechst-33342	Sigma, St. Louis, MO
Inverted fluorescent microscope	Leica, Buffalo Grove, IL	Leica DMI 6000B
Inverted fluorescent microscope	Olympus, Shinjuku, Japan	Olympus 1x70
Normal goat serum	Sigma, St. Louis, MO
Paraffin	Sigma, St. Louis, MO
Paraformaldehyde	Sigma, St. Louis, MO
Ploy-lysine coated charged glass slide	Globe Scientific Inc, Mahwah, NJ
Potassium chloride	Sigma, St. Louis, MO
Potassium dihydrogen phosphate	Sigma, St. Louis, MO
Sodium azide	Sigma, St. Louis, MO
Sodium chloride	Sigma, St. Louis, MO
Sodium hydroxide	EMD Millipore, Burlington, MA
Sodium pentobarbital	Vortex Pharmaceuticals limited, Dearborn, MI
Thioflavin-S	Sigma, St. Louis, MO
Triton-X-100	Sigma, St. Louis, MO
Xylene	VWR,Radnor, PA