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 thought to be involved in synaptic loss, increased neuroinflammation, and neurodegeneration in the cortical and hippocampal regions in brain affected by AD⁵. Therefore, histochemical detection of Aβ in AD tissue is a crucial first step in developing non-toxic, anti-amyloid drugs to prevent AD progression.

During the past few decades, several dyes and antibodies have been used by many research laboratories to label and image Aβ plaques in brain tissue, but some of these methods are time consuming and the dyes or antibodies used are expensive, requiring several accessory chemicals. Therefore, the development of an inexpensive means of detection of Aβ plaques in the AD brain would be a welcome new tool. Many laboratories started using Cur, a promising anti-amyloid natural polyphenol, for labeling and imaging Aβ, as well as a therapeutic agent for AD^6,7,8,9. Its hydrophobicity and lypophilic nature, structural similarities with classical amyloid binding dyes, strong fluorescent activity, as well as strong affinity to bind with Aβ makes it an ideal fluorophore for labeling and imaging of Aβ plaques in AD tissue¹⁰. Cur binds with Aβ-plaques and oligomers and its presence is also detected in intracellular spaces^7,11,12,13. In addition, it has been shown that minimal amounts (1−10 nM) of Cur can label Aβ plaques in 5x familial Alzheimer's disease (5xFAD) brain tissue⁷. Although the 1 nM concentration does not provide the optimal fluorescence intensity for counting of Aβ plaques, a 10 nM or higher concentration of Cur does. Ran and colleagues¹⁴ reported that doses as low as 0.2 nM of difluoroboron-derivatized Cur can detect in vivo Aβ deposits nearly as well as an infrared probe. Whether this dose is sufficient to label Aβ plaques in tissue is still not clear. Most previous studies have used 20−30 min for staining Aβ plaques using Cur, but optimal staining may require much less time.

The present study was designed to test the minimum time required by Cur to label Aβ plaques in AD brain tissue and to compare the sensitivity for labeling and imaging of Aβ plaques in brain tissue from the 5xFAD mice after staining with Cur with other conventional Aβ-binding dyes, such as Thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC). The Aβ labeling capability of these classical amyloid binding dyes was compared with Cur staining in paraffin-embedded and cryostat coronal brain sections from 5xFAD mice and from aged-matched human AD and control brain tissue. The findings suggest that Cur labels Aβ plaques in a manner similar to Aβ-specific antibodies (6E10) and moderately better than Thio-S, CR, or FJC. In addition, when intraperitoneal injections of Cur to 5xFAD mice were administered for 2−5 days, it crossed the blood-brain barrier and bound with Aβ plaques⁷. Interestingly, nanomolar concentrations of Cur have been used to label and image Aβ plaques in 5xFAD brain tissue^7,14. Moreover, morphologically distinct Aβ plaques, such as core, neuritic, diffuse, and burned-out plaques can be labeled by Cur more efficiently than with any of the other conventional amyloid binding dyes⁷. Overall, Cur can be applied to label and image Aβ plaques in postmortem brain tissue from AD animal models and/or human AD tissue in an easy and inexpensive way, as a reliable alternative to Aβ-specific antibodies.

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 (KH₂PO₄), and double distilled water to make a total of 1 L.
   2. Prepare 4% paraformaldehyde (PFA).
      1. Add 40 g of paraformaldehyde to 1 L of PBS (0.1 M, pH 7.4).
      2. Heat the PFA solution to 60−65 °C and mix using a magnetic stirrer.
         NOTE: The temperature should not exceed 65 °C.
      3. Add few drops of NaOH (1 N) with a dropper to dissolve the PFA completely.
      4. Filter the PFA solution with medium to fine filter paper and store at 4 °C.
         NOTE: The solution is good for a month.
2. Perform animal anesthesia and perfusion.
   NOTE: Twelve-month-old B6SJL-Tg APP SwFlLon, PSEN1*M146L*L286V, 1136799Vas/J (5×FAD) age-matched control mice (n = 6 per group) were purchased from vendors and bred in the animal house of Saginaw Valley State University. Genotyping was confirmed by polymerase chain reaction (PCR) as described previously⁷. Human AD brain tissue includes postmortem AD brain tissue and age-matched control tissue.
   1. Anesthetize the animal with an appropriate anesthetic agent, such as sodium pentobarbital (390 mg/kg body weight), or a ketamine/xylazine mixture (up to 80 mg/kg body weight ketamine and 10 mg/kg body weight xylazine) by intraperitoneal injection (27 G needle and 1 mL syringe). Check the level of anesthesia by pinching a toe. If the animal is unresponsive, then it is ready for perfusion surgery.
   2. Place anesthetized animal in the supine position on the perfusion surgery tray and using small iris scissors make an incision to the posterior end of the left ventricle.
   3. Insert a 22 G perfusion needle to the left ventricle and make a small incision at the right auricle to remove perfusion fluid from the body. Use a gravity-fed perfusion system to allow the ice-cold perfusion fluid (0.1 M PBS, pH 7.4) to flow for 5−6 min (flow rate 20−25 ml/min).
      NOTE: A clear liver is the indicator of optimum perfusion.
   4. Switch the buffer valve to an ice-cold 4% paraformaldehyde solution for fixing and allow it to flow for 8−10 min.
      NOTE: Tremor followed by hardened or stiff limbs are indicators of good fixation.
   5. Remove the brain from the skull using scissors. Using a spatula, collect the brain and place it in a vial of 4% PFA (at least 10x the volume of the brain volume) and store at 4 °C until further use.

2. Tissue processing

1. Cut cryostat sections.
   1. Transfer the brain to graded sucrose solutions (10%, 20%, and 30%) and store at 4 °C for 24 h each, until use.
   2. Using a cryostat at -22 °C, cut 40 µm-thick sections. Collect 10−20 sections per well in a 6 well plate filled with PBS and sodium azide (0.02%).
2. Paraffin embed the sections for mouse and human brain tissue.
   1. For paraffin sections, dehydrate the perfused and 24 h post-fixed brain tissue with graded alcohols (50%, 70%, 90%) for 2 h each, followed by 100% alcohol 2x for 1 h each), and then with xylene 2x for 1 h each) at room temperature.
   2. Penetrate the tissue with xylene-paraffin (1:1) 2x for 1 h at 56 °C in a glass conical flask covered with aluminum foil.
   3. Immerse the tissue in melted paraffin (56 °C) for 4−6 h.
   4. Cut 5 µm-thick sections using a rotary microtome at room temperature and place them in a tissue water bath at 45 °C.
3. Histochemically label the Aβ plaques in the cryostat sections with Cur.
   1. Rinse the sections from step 2.1.2 with PBS (pH 7.4) 3x for 5 min each.
   2. Immerse the sections in 70% ethanol for 2 min at room temperature.
   3. Dissolve stock Cur (1 mM) in methanol and dilute with 70% ethanol to obtain a final working concentration of 10 µM.
   4. Immerse the sections with working Cur solution for 1−5 min at room temperature on a shaker at 150 rpm.
   5. Discard Cur solution and wash with 70% ethanol 3x for 2 min each.
   6. Put the sections on poly-L-lysine coated glass slides and mount with a coverslip using organic mounting media, such as distyrene plasticizer xylene (DPX).
   7. View under a fluorescence microscope using 480/550 nm excitation/emission filters.
4. Histochemically label the Aβ plaques in the paraffin-embedded mouse and human brain sections with Cur.
   1. Deparaffinize the tissue sections from step 2.2.4 with xylene 2x for 5 min each at room temperature.
   2. Rehydrate with graded alcohol solutions (100%, 80%, 70%, 50% for 1 min each) and with distilled water 2x for 5 min each at room temperature.
   3. Stain sections with Cur (10 µM) for 10 min at room temperature in the dark, shaking at 150 rpm. Wash with 70%, 90%, and 100% alcohol for 2 min each.
   4. Clear with xylene 2x for 5 min each and cover slip with DPX.
   5. Visualize under a fluorescence microscope as mentioned in step 2.3.7.
5. Colocalize Cur with the Aβ antibody in Aβ plaques and oligomers.
   1. Wash cryostat sections from step 2.1.2 with PBS 3x in a 12 well plate.
   2. Block the sections with 10% normal goat serum (NGS) dissolved in PBS with 0.5% Triton-X-100 at room temperature for 1 h.
   3. Discard the blocking solution. Incubate the sections with Aβ-specific antibodies (6E10 or A11, diluted 1:200) dissolved in fresh blocking solution containing 10% NGS and 0.5% Triton-X100 overnight at 4 °C in a shaker at 150 rpm.
   4. Discard the antibody solution and wash the sections with PBS 3x for 10 min each.
   5. Incubate with the secondary antibody tag with red fluorophore (e.g., Alexa 594) for 1 h at room temperature in the dark.
   6. Wash with PBS 3x for 10 min each.
   7. Wash with 70% alcohol 1x.
   8. Incubate the sections with Cur (10 µM) for 5 min at room temperature.
   9. Wash with 70% alcohol 3x for 1 min each.
   10. Dehydrate with 90% and 100% alcohol for 1 min each, clear with xylene 2x for 5 min each, and mount on slides using DPX.
   11. Visualize using a fluorescence microscope with appropriate excitation/emission filters for the red and green signals.
   12. For intracellular Aβ colocalization, stain the sections using Aβ-antibody (6E10), restain with Cur at room temperature in the dark with shaking at 150 rpm, and counterstain with either Hoechst-33342 (1 mg/ml) and/or DAPI (1ug/ml) for 10 min at room temperature in the dark with shaking at 150 rpm. Wash with PBS 3x.
   13. Take images with the red, green, and blue filters with a 100x objective (total magnification 1,000x).
6. Label Aβ plaques with Thio-S, CR, and FJC.
   NOTE: Detailed protocols for Thio-S and CR labeling were previously reported⁷.
   1. For FJC staining, wash the free-floating sections obtained from step 2.1.2 with PBS 3x for 5 min each.
   2. Place the sections in a 12 well plate and stain with FJC (0.001%) for 10 min in the dark at room temperature.
   3. Discard FJC solution and wash with PBS 3x for 5 min each.
   4. Incubate with ammonium chloride (NH₄Cl, 50 mM dissolved in PBS) for 10 min at room temperature.
   5. Discard the NH₄Cl solution and wash with PBS 3x for 5 min each.
   6. Following the steps in section 2.4, dehydrate with graded alcohol solutions, clear, mount, and view under a fluorescence microscope using 450/520 nm excitation/emission filters.

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-prepare...

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

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