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Histochem Cell BiolDOI 10.1007/s00418-016-1464-1

ORIGINAL PAPER

A comparative study of dietary curcumin, nanocurcumin, and other classical amyloid‑binding dyes for labeling and imaging of amyloid plaques in brain tissue of 5×‑familial Alzheimer’s disease mice

Panchanan Maiti1,2,3,5 · Tia C. Hall1,2 · Leela Paladugu1,2 · Nivya Kolli1,2 · Cameron Learman1,2 · Julien Rossignol1,2,4 · Gary L. Dunbar1,2,3,5

Accepted: 24 June 2016 © Springer-Verlag Berlin Heidelberg 2016

antibody and to a greater extent than those of the classical amyloid-binding dyes. Cur and NC also labeled Aβ plaques in 5×FAD brain tissues when injected intraperitoneally. Nanomolar concentrations of Cur or NC are sufficient for labeling and imaging of Aβ plaques in 5×FAD brain tis-sue. Cur and NC also labeled different types of Aβ plaques, including core, neuritic, diffuse, and burned-out, to a greater degree than other amyloid-binding dyes. Therefore, Cur and or NC can be used as an alternative to Aβ-specific antibody for labeling and imaging of Aβ plaques ex vivo and in vivo. It can provide an easy and inexpensive means of detecting Aβ-plaque load in postmortem brain tissue of animal models of AD after anti-amyloid therapy.

Keywords Curcumin · Nanocurcumin · Protein labelling · Alzheimer’s disease · Amyloid beta protein

AbbreviationsCur CurcuminNC NanocurcuminAPP Amyloid precursor proteinAβ Amyloid beta proteinPET Positron emission tomographyAD Alzheimer’s diseaseThio-S Thioflavin-SCR Congo red5× FAD Five times familiar Alzheimer’s diseaseDMSO Dimethyl sulfoxidePBS Phosphate buffer salineABC Avidin biotin complexDAB DiaminobenzidinePD Parkinson’s diseaseHD Huntington’s diseaseAU Arbitrary unitHSD Honestly significant difference

Abstract Deposition of amyloid beta protein (Aβ) is a key component in the pathogenesis of Alzheimer’s disease (AD). As an anti-amyloid natural polyphenol, curcumin (Cur) has been used as a therapy for AD. Its fluorescent activity, preferential binding to Aβ, as well as structural similarities with other traditional amyloid-binding dyes, make it a promising candidate for labeling and imaging of Aβ plaques in vivo. The present study was designed to test whether dietary Cur and nanocurcumin (NC) provide more sensitivity for labeling and imaging of Aβ plaques in brain tissues from the 5×-familial AD (5×FAD) mice than the classical Aβ-binding dyes, such as Congo red and Thiofla-vin-S. These comparisons were made in postmortem brain tissues from the 5×FAD mice. We observed that Cur and NC labeled Aβ plaques to the same degree as Aβ-specific

Electronic supplementary material The online version of this article (doi:10.1007/s00418-016-1464-1) contains supplementary material, which is available to authorized users.

* Panchanan Maiti maiti1p@cmich.edu

* Gary L. Dunbar dunba1g@cmich.edu

1 Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mt. Pleasant, MI 48859, USA

2 Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA

3 Department of Psychology, Central Michigan University, Mt. Pleasant, MI 48859, USA

4 College of Medicine, Central Michigan University, Mt. Pleasant, MI 48859, USA

5 Field Neurosciences Institute, St. Mary’s of Michigan, Saginaw, MI 48604, USA

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Introduction

Gradual accumulations of denatured, misfolded, or mutated amyloid beta proteins (Aβ) inside and outside of neurons are the key pathological hallmarks of Alzheimer’s disease (AD) (Harrison et al. 2007; Merlini and Westermark 2004). Aggregations of this Aβ causes the progressive loss of neuronal communication, synaptic integrity, and neuronal regeneration contributing to pathogenesis of AD, for which a cure or an effective treatment is still lacking (Selkoe 2004; Serrano-Pozo et al. 2011). Recently, as a potent anti-amyloid natural polyphenol, curcumin (Cur) has become one of the most promising compounds for inhibition of misfolded Aβ aggregation (Hu et al. 2015; Lim et al. 2001; Yang et al. 2005).

Curcumin is a bright, yellow-colored pigment, derived from the root of the herb, Curcuma longa, a traditional die-tary spice, and is considered one of the “magic molecules” in Indian and South Asian Ayurvedic medicine (Prasad and Aggarwal 2011). Because of its pleiotropic actions, such as anti-amyloid (Ono et al. 2004; Yang et al. 2005), anti-oxidant (Menon and Sudheer 2007), and anti-inflammatory properties (Ray and Lahiri 2009), Cur has been targeted for therapeutic application in several brain diseases (Frautschy and Cole 2010; Hu et al. 2015; McClure et al. 2015). It can bind to senile plaques and is thought to decrease Aβ depos-its, in vivo (Hu et al. 2015; Lim et al. 2001; Ma et al. 2013; Ono et al. 2004; Yang et al. 2005). Further, Cur treatment also decreases Aβ plaques in both the amyloid - precursor- protein (APP) and tau - transgenic mouse models of AD (Garcia-Alloza et al. 2007; Ma et al. 2013).

Interestingly, Cur has specificity for Aβ plaques and high-affinity binding for Aβ (Kd = 0.20 nM); (Ran et al. 2009; Yang et al. 2005). Because of its fluorescent proper-ties, researchers have tried to use Cur derivatives for in vivo imaging, such as potential positron emission tomographic (PET) probes for amyloid imaging or retinal scans for detection of AD in experimental animals and humans (Ryu et al. 2006). Similarly, a probe derived from boro-fluoro-Cur has been shown to have several times higher fluores-cence properties than natural Cur upon binding to Aβ pro-teins (Ran et al. 2009; Zhang et al. 2014). Furthermore, when the brain sections from AD patients and animal mod-els were incubated with Cur, a strong fluorescent signal was observed, indicating that Cur has preferential binding to Aβ plaques (Koronyo et al. 2012; Zhang et al. 2015a).

However, the poor solubility, instability in physiological fluids, and low bioavailability of Cur are the major obsta-cles for delivering it in therapeutically significant amounts for treating various neurological diseases (Anand et al. 2007; Kumar et al. 2010). Cur provides a weak fluorescent signal in water, but has greater fluorescent activity in lipid or hydrophobic environments (Kunwar et al. 2006). The

hydrophobic nature within the core of Aβ-plaques makes it easier to interact with Cur, producing intense fluorescent activity. Several investigators have tried to dissolve Cur in different lipid formulas, including the use of solid lipid nanoparticles (Ghalandarlaki et al. 2014). Recently, a solid lipid nanoparticle (SLNP) formulation, conjugated with Cur (nanocurcumin; NC), has been shown to increase its solubility, stability, and bioavailability and confers greater neuroprotection in mouse models of AD (Begum et al. 2008; Frautschy and Cole 2010; Ma et al. 2013; Hu et al. 2015). This SLNP-Cur formula is considered a promising strategy for restoration and up-regulation of several syn-aptic markers and for protein clearance pathways in trans-genic mouse models of AD (Begum et al. 2008; Ma et al. 2013; Maiti et al. 2014; Hu et al. 2015). This formulation has 65 times the penetrance into the brain tissue and can bind to Aβ-plaques more effectively than dietary Cur, a finding which may lead to mitigating the deleterious effects of misfolded protein aggregates in several neurodegenera-tive diseases (Begum et al. 2008; Ma et al. 2013; Maiti et al. 2014; Hu et al. 2015).

Several reports are available regarding the localization of Aβ plaques in tissue using different classical amyloid-binding dyes, such as Thioflavin-S (Thio-S) and Congo red (CR); (Bussiere et al. 2004; Maezawa et al. 2008). Whereas Cur has structural similarities to these classical amyloid-binding dyes (Fig. 1), the use of these compounds to label and image Aβ plaques in vivo and postmortem brain tissue from 5×FAD has not been adequately addressed. Moreo-ver, the solid lipid nanoparticle formulation of NC may have greater binding capability to the hydrophobic core of Aβ plaques than does dietary Cur or other compounds of turmeric extract. Given this, the present study was designed to compare the labeling and imaging capability of dietary Cur, NC, and other traditional amyloid-binding dyes with Aβ-specific antibody on postmortem and in vivo brain tis-sue of a genetically modified mouse model of familial AD (5×FAD). Our results suggest that the dietary Cur or NC can be used as an alternative of Aβ-specific antibody, which allows a quick and easy way to look for Aβ-plaque load in postmortem and in vivo brain tissue after anti-amyloid ther-apy in experimental models of AD.

Materials and methods

Chemicals

Thio-S (practical grade), Congo red, curcumin (Cur, ~80 % pure), and other accessory chemicals were pro-cured from Sigma (St. Louis, MO). Aβ antibody (6E10) was purchased from BioLegend (San Diego, CA), and Aβ-oligomer-specific antibody (A11) was procured from

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Millipore (Billerica, MA). Solid lipid nanoparticle contain-ing Cur (Nanocurcumin: NC, also called Longvida, which contains 20 % pure Cur) was a kind gift from Verdure Sci-ence (Noblesville, IN). This NC consists of high-purity, long-chain phospholipid bilayer and a long-chain fatty acid solid lipid core, which coats the Cur. The NC has been well characterized by the Cole and Frautschy laboratory, at the University of California, Los Angeles, in collaboration with Verdure Science (Cox et al. 2015; DiSilvestro et al. 2012; Ma et al. 2013; Nahar et al. 2015). Cur-extract and NC con-tain 80 and 20 % of pure Cur, respectively. However, each selected concentration (1 mM, 100, 10, 1 μM, 100, 10, and 1 nM) used in the present study was based on the actual amount of Cur or NC found in their respective extracts.

Animals

One-year-old B6SJL-Tg (APPSwFlLon, PSEN1*M146L* L286V, 1136799Vas/J; also called 5×FAD, Jackson Laboratory) mice (n = 9) were used for this study. These mice overexpress human APP and PS1 with five famil-ial AD mutations, including three mutations on APP gene [Swedish (K670N, M671L), Florida (I716V), and Lon-don (V717I)] and two on PS1 gene (M146L and L286V) (Maya-Vetencourt et al. 2014; Xu et al. 2014). These mice develop Aβ plaques in selected regions of the brain, one

of the hallmark pathologies of AD, along with concomi-tant behavioral deficits, including memory loss and cog-nitive impairments at about 2 months of age (Kimura and Ohno 2009; Oakley et al. 2006; Ohno et al. 2006). The 5×FAD mice were housed at 22 °C under a 12-h light/12-h dark, reverse-light cycle with ad libitum access to food and water. All mice were genotyped at 3 weeks of age by polymerase chain reaction (PCR) to confirm their trans-genic characteristics, as reported previously (Sadleir et al. 2015). This study was carried out in strict accordance with the protocols approved by the Institutional Animal Care and Use Committee of the Central Michigan University (IACUC 09-13A). All procedures were performed under proper anesthetic conditions, and all efforts were made to minimize animal suffering.

Solubility of Cur and NC

To assess their solubility, different concentrations of Cur and NC were dissolved in different solvents, such as NaOH (10 N), DMSO, methanol, and PBS, then vortexed vigor-ously and allowed to sit for 30 min at room temperature in the dark. Ten microliters of the solution was then put onto a glass slide, cover-slipped, and visualized using a fluores-cence microscope (Leica, Germany), with a green filters (excitation/emission:480/550).

Fig. 1 Comparison of Cur, NC with classical amyloid-binding dyes. Different parameters, including their chemical structure, physical appear-ance, binding to Aβ, and possible interactions with Aβ were compared with Cur, NC, Thio-S, and CR

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Injection of Cur and NC to 5×FAD mice

Age-matched wild-type and 5×FAD mice (1 years old, n = 3/group) were injected with Cur and NC to check their Aβ-labeling capability in vivo. Briefly, Cur and NC were dissolved in methanol and diluted in 0.1 mM PBS (pH 7.4) and injected intraperitoneally (one injection per day) into mice at the dose of 50 mg/kg body weight for 2 and 5 days.

Tissue processing

All mice were deeply anaesthetized with an overdose of sodium pentobarbital (intraperitoneally) and transcardially perfused with 0.1 M PBS, followed by 4 % paraformalde-hyde (diluted in 0.1 M PBS at pH 7.4) to fix the brains. The brains were then rapidly removed, suspended in 4 % para-formaldehyde for 24 h at 4 °C, then transferred to graded sucrose solutions (10, 20, and 30 %) dissolved in 0.1 M PBS, and stored at 4 °C, until the brain was completely immersed in the solution. For paraffin sections, the tissues were dehydrated by graded alcohol (50, 70, 90 %) for 2 h each and 100 % alcohol (1 h for two changes), followed by xylene (1 h, two changes), then impregnated with xylene-paraffin (1:1) overnight, and then immersed in melted par-affin for 4 h. The paraffin block was sectioned with a rotary microtome at 5 μm thickness.

Aβ plaque‑imaging after intraperitoneal injection of Cur and NC

Within 2 h after the stipulated doses of Cur and NC were injected intraperitoneally, the mice were killed and their brain tissue was processed as described above. Coronal sections (40 μM) were counterstained with propidium iodide and the Aβ plaques stained by Cur and NC were imaged using a fluorescent microscope (Leica, Germany) with appropriate filters.

Histochemical labeling of Aβ plaques by Cur and NC

Histochemical labeling of Aβ plaques with Cur and NC was performed as described previously (Garcia-Alloza et al. 2007; Koronyo et al. 2012; Mutsuga et al. 2012). Briefly, perfused brains were cryopreserved and coronal sections (30 μm thickness) were obtained using a cryostat (Leica, Germany). The sections were rinsed three times with 1 mM PBS (pH 7.4) for 5 min each. Then the sec-tions were stained with different concentrations of Cur and NC (1 mM, 100, 10, 1 μM, 100, 10, and 1 nM, dis-solved in methanol) for 30 min at room temperature on a shaker (at 150 rpm). The sections were thoroughly washed 3 times for 5 min each, air-dried, and mounted with anti-fading Fluoro-mount aqueous mounting media (Sigma) and

visualized using a fluorescence microscope (Leica, Ger-many), using 480/550 nm excitation/emission filters. For paraffin sections, the tissue was deparaffinized with xylene (two changes, 5 min each), followed by rehydration with graded alcohol solutions (100, 80, 70, 50 % for 1 min each) and with distilled water for 5 min (two changes). Then the sections were stained with Cur/NC (1 μM), as described above, and the photomicrographs were taken using the same microscope as mentioned above.

Quantification of fluorescent intensity of Aβ plaques stained by different concentrations of Cur and NC

The fluorescent intensity (arbitrary unit, AU) of Aβ-stained plaques by different concentrations of Cur and NC was measured using ImageJ software (http://imagej.nih.gov/ij). Briefly, 40-µm-thick sections were stained with differ-ent concentrations of Cur and NC (1 mM, 100, 10, 1 μM, 100, 10, and 1 nM, dissolved in methanol) and the images were taken as described previously. The fluorescent inten-sity of each individual Aβ plaque was measured manually using ImageJ software (http://imagej.nih.gov/ij). About 100 Aβ plaques in each group from three different mice were measured and averaged. Control tissue was used to measure the background signal after staining, and the background signal was subtracted from the fluorescent signal obtained from the experimental tissues.

Congo red staining

Labeling of Aβ plaques with CR was performed as described previously (Mutsuga et al. 2012; Tei et al. 2012). Briefly, the cryostat sections (15 μm) were washed three times with 1 mM PBS (pH 7.4) and rehydrated under run-ning lukewarm tap water for 20–30 min. The sections were stained with different concentrations of CR solu-tion, such as 1, 0.5, 0.25, and 0.125 % dissolved in 80 % ethanol along with 1 % NaOH for 1 h at room tempera-ture, followed by a rinse in running, lukewarm tap water for 15–20 min. Then the sections were counterstained with 0.1 % Cresyl violet (Acros Organic, NJ) for 2 min and washed three times with distilled water, dehydrated through an ascending alcohol series (70, 90, and 100 %), and then placed into xylene and cover-slipped with DPX (Electron Microscopic Sciences, Washington, PA). The sections were visualized using a compound light micro-scope (Olympus, Japan) or with a polarized microscope (Leica, Germany).

Thioflavin‑S (Thio‑S) staining

The Thio-S staining procedure was followed as described previously (Bussiere et al. 2004; Sun et al. 2002). Briefly,

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Thio-S solution at 1 % (w/v) was prepared by dissolving 1 g of Thioflavin-S (Sigma-Aldrich) in 80 % ethanol (v/v). The tissue sections were first washed with PBS, followed by graded alcohol (50, 70, and 80 %) for one min each. The sections were incubated with 0.1 % Thio-S for 10 min, dipping them three times in 70 % ethanol (v/v), and finally washed once in PBS, before being mounted, as described above. The sections were visualized using a fluorescence microscope (Leica, Germany), with excitation/emission fil-ters (480/550 nm).

Aβ immunohistochemistry

Aβ immunohistochemistry was performed using both the immunofluorescent and immunoperoxidase techniques. Briefly, after cryopreservation, 15-μm-thick sections were obtained using a cryostat (Leica, Germany). The sections were rinsed with PBS (0.1 mM, at pH 7.4) three times and then incubated with 0.5 % Triton-X100 (Fisher Sci-entific, Pittsburgh, PA), along with 0.3 % H2O2 solution (for immune-peroxidase technique), for 30 min at room temperature, followed by three washes in PBS, for 10 min each. The unmasking was done by treating the sections with 10 % normal goat serum (Santa Cruz Biotech, Dallas, Texas) for 1 h at room temperature. Then sections were incubated with mouse monoclonal anti-Aβ-antibodies (6E10; 1:200; A11; 1:1000), which were dissolved in PBS, along with 10 % goat serum and placed on a shaker at low speed and kept at 4 °C overnight. On the next day, the sec-tions were thoroughly washed with PBS, three times for 10 min each. For the immunofluorescent protocol, the sections were incubated with anti-mouse secondary anti-body (1:200), tagged with Alexa-594 (Molecular Probes, OR) for 30 min at room temperature. Then the sections were washed thoroughly with distilled water, dehydrated, cleared, and mounted on slides using anti-fading Fluoro-mount aqueous mounting media (Sigma), and visualized using a fluorescence microscope (Leica, Germany) with appropriate excitation and emission filters. Similarly, for the immunoperoxidase protocol, the tissue was thoroughly washed with PBS after it had been incubated overnight at 4 °C with primary antibody. The tissue was then incubated with biotinylated anti-mouse secondary antibody (Vector Laboratory, CA; 1:200) for 30 min at room temperature. After this incubation, the sections were washed three times with PBS, 10 min each, and then treated with ABC rea-gent for 30 min at room temperature. This was followed by three washes in PBS of 10 min each. Finally, the sec-tions were incubated with peroxidase substrate solution, supplied with the ABC kit (Vector Laboratory, CA), and the signal was developed using diaminobenzidine (DAB) until the desired staining intensity emerged. The tissue was then washed, dehydrated, cleared, mounted on slides, and

visualized using a compound light microscope (Olympus, Japan).

Co‑localization of Cur and NC with Aβ antibody (6E10) in Aβ plaques

After Aβ immunostaining, some of the sections were stained with Cur and some of them with NC, in order to quantify their co-localization with Aβ antibody in Aβ plaques. Briefly, after immunofluorescent or immunop-eroxidase staining (as described above), the sections were stained with Cur and NC (1 µM) for 30 min at room tem-perature and then washed thoroughly with PBS for three times, 10 min each. Then, the sections were washed with distilled water, dehydrated, cleared, and mounted on slides using anti-fading Fluoro-mount aqueous mounting media (Sigma) and visualized using a fluorescence microscope (Leica, Germany), with appropriate excitation/emission fil-ters. For intracellular Aβ co-localization, the sections were stained using Aβ antibody (6E10) and Cur or NC, the sec-tions were counterstained with Hoechst-33342 for 10 min and images were taken using the same microscope with red/green/blue filters at 100 × objective (total magnifica-tion 1000×).

Counting the number of Aβ plaques

For quantification of Aβ plaques, thin cryostat sections (15 µm thickness) were used to visualize all plaques in a single focal point in order to avoid biased quantification. Briefly, the images were taken from randomly selected regions of the layer IV/V of prefrontal cortex area and also from dentate gyrus of the hippocampus using 20× objec-tives (total magnification 200 times). Using ImageJ soft-ware (http://imagej.nih.gov/ij), the total area of each image was measured and the number of Aβ plaques were counted manually in that area. Only clearly visible large fluorescent (in case of Cur and NC) and reddish-pink signals (in case of CR in compound light microscope) were considered as Aβ plaques, and small fluorescent dots were eliminated from the count. A number of Aβ plaques were expressed per 100 µm2 area. To count the number of Aβ plaques in each group, a minimum of 10 serial sections, each with a hundred different fields, were sampled. Samples from three different animals from each group were used, with the experimenter blinded to the group identity of the specimens.

Statistical analysis

The morphometric data for Aβ-plaque counts and the flu-orescent intensity of Aβ plaques, at different time points, were expressed as mean ± SEM. Data were analyzed using one-way analysis of variance (ANOVA) with Tukey HSD

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(honestly significant difference) post hoc tests being con-ducted, when appropriate. A probability value below or equal to 0.05 was considered as statistically significant.

Results

Solubility of Cur and NC

To compare the solubility of Cur and NC, these compounds were dissolved in different solvents at different concentra-tions (100–0.001 µM). With Cur, large fluorescence crys-tals were observed in all the mentioned solvents, indicating dietary Cur did not completely dissolve in NaOH (10 N), DMSO, methanol, or PBS, as expected. However, the high-est degree of solubility for Cur was observed in methanol, compared to all of the other solvents. Unlike Cur, large flu-orescence crystals were not observed when NC was placed in all of these solvents, indicating it was solubilized by most of the solvents, except that some small particles were noticed in the DMSO and PBS (S1‑A). Cur also showed rapid degradation in PBS (0.1 M, pH 7.4), compared to NC, as shown by its fluorescent intensity (S1‑B).

Labeling of Aβ plaques by Cur and NC was similar to that of Aβ antibody

The 5×FAD brain sections (cryo-sections) were stained with dietary Cur and NC (1 µM), and Aβ-specific antibody (6E10), and after binding to Aβ plaques, intense fluores-cence signals were observed throughout the tissue of the Cur/NC-stained sections, which appeared as intense as those sections stained by Aβ-specific antibody (Fig. 2). In addition, in many experimental studies, Aβ immunohisto-chemistry is performed using paraffin-embedded sections. Therefore, to test whether Cur or NC can stain Aβ plaques in paraffin sections as well as frozen sections, some par-affin sections (5 µm) also were stained with same concen-tration (1 µM) of Cur/NC. We observed that Cur/NC also labeled Aβ plaques in paraffin sections, at intensity levels equivalent to what is observed in frozen sections (Fig. 2).

Co‑localization of Cur and NC with Aβ‑specific antibody (6E10)

To confirm whether the fluorescent signals were due to binding of Cur with Aβ plaques or whether it was due to

Fig. 2 Labeling of Aβ plaques with Cur, NC, and Aβ-antibody. 5×FAD cryostat and paraffin-embedded sections were stained by dietary Cur or NC (1 µM) and with Aβ antibody (6E10). The color of Aβ-antibody labeled sections were developed by biotinylated sec-ondary antibody followed by diaminobenzidine. Note that Cur and

NC labeled Aβ plaques in a manner similar to Aβ-specific antibody (6E10) in both frozen and paraffin-fixed tissue. Arrows indicate Aβ-plaques. Scale bar indicates 100 μm and is applicable to all other images

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binding somewhere else, Aβ plaques were labeled with Aβ-specific mouse monoclonal antibody (6E10) and then the same sections were stained with Cur and or NC. Nearly 100 % of all plaques were stained with Cur and NC, and these co-localized with Aβ antibody (Fig. 3).

Dietary Cur and NC labeled Aβ oligomers to a similar extent as did the Aβ‑oligomer‑specific antibody (A11)

To check whether Cur and NC bind to Aβ oligomers, we stained postmortem 5×FAD brain sections first with Aβ-oligomer-specific antibody (A11) followed by Cur and NC (1 µM). We observed that Cur and NC completely co-localized with Aβ oligomers, where A11 binds (Fig. 4).

Dietary Cur and NC labeled intracellular Aβ to a similar as did the Aβ‑specific antibody (6E10)

To check whether Cur and NC can label intracellular Aβ, as well as extracellular Aβ plaques, higher magnified images (1000×) were taken from the sections which were stained with 6E10 followed by Cur and NC. We observed that Cur or NC also co-localized with Aβ antibody (6E10) in intra-cellular spaces, indicating Cur and NC can also label intra-cellular Aβ (Fig. 5).

Dietary Cur and NC labeled Aβ plaques in vivo

To check labeling capability of Cur and NC in vivo, the 5×FAD mice were intraperitoneally injected with Cur or NC at different time points. We found that both Cur and NC crossed blood brain barrier and bound with Aβ plaques

(Fig. 6a) in 5×FAD mouse brain tissue, whereas in the brain sections of age-matched wild-type mice, we did not observe any fluorescent signals. In addition, NC showed more labeling of Aβ plaques than did Cur alone, at both 2 and 5 days following injections. Moreover, the labeling of either Cur or NC was even more intense at 5 days than after 2 days following injections (Fig. 6a). To confirm Cur or NC bind with plaques specifically, 5×FAD mice sections treated with Cur or NC were immunolabeled by Aβ-specific antibody (6E10). We observed that Cur or NC completely co-localized with Aβ-specific antibody, suggesting Cur or NC specifically bind with Aβ plaques, as observed in post-mortem 5×FAD brain tissue (Fig. 6b).

Dietary Cur and NC labeled Aβ plaques with greater sensitivity than classical amyloid‑binding dyes

When we compared labeling of Aβ plaques with Cur, NC, and other classical amyloid-binding dyes, such as Thio-S, CR, and also with Aβ-antibodies, greater plaque labeling by both Cur and NC was observed, compared to labeling by Thio-S and CR (Fig. 7). The Cur and NC had a simi-lar binding capacity for Aβ plaques as those labeled by Aβ antibody (Fig. 7a). The mean number of plaques labeled by Cur (13.18/100 µm2) and NC (14.06/100 µm2), which were similar to Aβ antibody (13.52/100 µm2), and significantly higher than Thio-S (10.99/100 µm2; p < 0.05) and CR (6.58/100 µm2; p < 0.01; Fig. 7b), when measured from over 100 areas in the prefrontal cortex of the 5×FAD mouse brain tissue. A similar finding was also observed in the case of dentate gyrus of hippocampus (Fig. 7c; p < 0.01).

Fig. 3 Co-localization of Cur and NC with Aβ antibody in Aβ plaques. 5×FAD sections were first labeled with Aβ antibody (6E10), followed by staining with Cur and or NC. Red 6E10 bound by secondary antibody tagged with Alexa fluorophore 594; green Cur or NC; black 6E10 bound by biotinylated secondary antibody and developed by ABC kit with DAB as substrate (in green filter). Note

that the Cur and NC were completely co-localized with Aβ, at the same plaque, where 6E10 binds. Arrows indicate Aβ plaques. Scale bar indicates 100 μm (40× objectives) which is applicable to all six images on the left, and 20 μm (100× objectives) which is applicable to all four images on the right

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Fig. 4 Co-localization of Cur and NC with Aβ-oligomer-specific antibody (A11) in Aβ. 5×FAD sections were first labeled with Aβ - oligomer-specific antibody (A11), followed by staining with Cur and or NC. Red A11 bound by secondary antibody tagged with Alexa

fluorophore 594; green: Cur or NC. Note that the Cur and NC were completely co-localized with Aβ oligomers, in the areas where A11 binds. Arrows indicate Aβ oligomers. Scale bar indicates 250 μm and is applicable to all other images

Fig. 5 Co-localization of intracellular Aβ with Cur or NC and Aβ antibody (6E10). Sections from 5×FAD mice were first labeled with Aβ antibody (6E10) followed staining them with Cur and or NC and then staining them with by Hoechst 33342 (nuclear stain). Red dots 6E10 bound to intracellular Aβ (arrow); green dots Cur or NC bound

to intracellular Aβ (arrow); blue Hoechst 33342. In merge images; arrows indicate intracellular Aβ co-localized with 6E10 and Cur or NC. Asterisks (*) indicate Aβ-plaques. Bar indicates 50 μm and is applicable to all other images

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Morphological characterization of Aβ plaques by dietary Cur, NC, and other amyloid‑binding dyes

To compare morphology of different Aβ plaques labeled by Cur, NC, and other classical amyloid-binding dyes, some of the 5×FAD sections were stained with all these compounds and photomicrographic images were taken at 40× objectives (Olympus, Japan) using GFP filter (total magnification 400×). Both Cur and NC labeled the core (radiating material), neuritic (a central dense core sur-rounded by either a corona of fibril-like material), diffuse (mesh of stained fibrils), and burned-out plaques (without any surrounding materials), in a manner similar to Thio-S, as reported previously (Bussiere et al. 2004), but Thio-S labeled fewer diffuse plaques (1 plaque/5 microscopic fields, 20× objective) in comparison with Cur and NC. Noticeably, CR failed to show such distinct morphologi-cal features, especially labeling of the neuritic and diffuse plaques, which can be clearly observed by Cur and NC

(Fig. 8), whereas, Aβ antibody (6E10) showed all types of Aβ plaques, except the neuritic ones, which were not as clear (a distinct core surrounded by a thin layer), as those observed in Cur- and NC-stained sections. In addition, the intensity of these types of plaques was greater when labeled by Cur and NC, compared to Thio-S. This discrepancy may be due to concentration of Thio-S (1 % w/v) used in this study, which was different from the concentrations of Cur or NC (1 μM).

Nanomolar concentration of Cur and NC labeled Aβ plaques

To determine the minimum concentration of Cur and NC required to label Aβ plaques in tissue, 5×FAD tissue sections were incubated with different concentrations (1 mM, 100, 10, 1 μM, 100, 10, and 1 nM) of Cur and or NC. Noticeably, both Cur and NC were able to label the plaques, even at the lowest, 1 nM concentration (Fig. 9).

Fig. 6 In vivo labeling of Aβ plaques with Cur and NC. Age-matched wild-type and 5×FAD mice (1 year old) were given intra-peritoneal injections of Cur or NC (50 mg/kg body weight) for 2 and 5 days. After treatments, the mice were killed and perfused by 4 % paraformaldehyde and cryoprotected with graded sucrose solu-tions before these brains were sectioned coronally (40 μm) and counterstained with propidium iodide. a Cur and NC crossed blood

brain barrier and bound with Aβ (green). After day 5, Aβ-labeling was greater than what was observed on day 2 for the Cur- and NC-injected mice and NC labeling of Aβ plaques was stronger than that of dietary Cur. b Co-localization of the NC with Aβ-specific antibody (6E10). Arrows indicate Aβ plaques. Bar indicates 250 μm and is applicable to all other images

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When we measured the fluorescent intensity (AU) of Aβ labeling by different concentrations of Cur and NC, the intensities were significantly lower in both Cur and NC at 1 nM level (p < 0.01) compared to other concen-trations (Graph A and B) and NC showed significantly greater intensity at 100 nM and 10 nM in comparison with Cur.

Labeling of Aβ plaques by different concentrations of Congo red solution

To investigate at what concentration of CR solution can optimally label Aβ plaques, we stained 5×FAD sections with different concentrations of CR solution (1, 0.5, 0.25, and 0.125 %) and counted the number of plaques in each concentration. We observed that CR solution contain-ing 0.25 % labeled Aβ plaques as effectively as solutions of 1 and 0.5 % (Fig. 10), whereas 0.125 % of CR solution labeled significantly fewer Aβ plaques in comparison with the other concentrations (p < 0.01).

Discussion

Because of its unique physical, chemical, and biological properties, including a potent anti-amyloid activity, Cur has promising therapeutic value for several neurological diseases (Lim et al. 2001; Garcia-Alloza et al. 2007; Hu et al. 2015). It has attracted much attention of researchers because of its low price and low toxicity. Our hypothesis was that both dietary Cur and NC might have greater bind-ing capability to Aβ plaques compared to classical amy-loid-binding dyes, at levels that would be similar to those observed using Aβ antibody. The aims of this study were to: (1) determine the minimum amount of Cur that is nec-essary to label and image Aβ plaques in postmortem brain tissue; (2) test whether NC has greater stability, solubility, bioavailability, and whether it may have enhanced capabili-ties for labeling Aβ plaques, relative to dietary Cur; (3) test whether NC labels Aβ plaques greater than Cur in vivo; and (4) determine whether Cur and/or NC can be used as an alternative to Aβ antibody for labeling Aβ plaques,

Fig. 7 Labeling of Aβ plaques with Cur, NC, and other classical amyloid-binding dyes. a Sections from 5×FAD mouse brains were labeled with NC, Cur, Thio-S, CR, and Aβ antibody (6E10). A num-ber of Aβ plaques labeled by Cur and NC at layer IV/V of cortex (b) and dentate gyrus of hippocampus (c) were similar to those labeled

with Aβ antibody and significantly higher than those labeled with Thio-S and CR. Arrows indicate Aβ plaque; *p < 0.05 compared to NC, **p < 0.01 compared to Cur, NC, Thio-S, and 6E10; ##p < 0.01 compared to Cur, NC, CR, and 6E10; scale bar indicates 250 μm and is applicable to all images

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while investigating amyloid plaque load after treatment with drugs for AD, as a less expensive alternative to using Aβ antibody. To this end, we compared Aβ-binding capa-bility of dietary Cur, NC, and different compounds present in turmeric extract with Aβ antibody with other classical amyloid-binding dyes, such as Thio-S and CR. Our obser-vations suggest that both dietary Cur and NC have bind-ing capability similar to Aβ antibody and have greater Aβ-labeling capability than classical amyloid-binding dyes.

Natural Cur may be less useful for treating neurodegen-erative diseases, due to its low solubility in body fluids, rapid degradation after intestinal absorption, and its limited bioavailability (Kumar et al. 2010; Hu et al. 2015). How-ever, when special, solid lipid nanoparticles are combined with Cur to make NC, this new compound is readily solu-ble in most of the solvents, even in the PBS (S1). There have been few reports available which described the Cur binding to Aβ plaques (Ono et al. 2004; Yang et al. 2005),

but none of them show how it can be used as a tool for labeling Aβ plaques, and even less information is available for comparative binding capability of dietary Cur, NC, and other classical amyloid-binding dyes (Mutsuga et al. 2012; Tei et al. 2012).

However, in the present study, we have investigated the Aβ-binding capability of Thio-S, a classical amyloid-binding dye, which has been used for the last few years to characterize Aβ aggregates histochemically. It binds with cross β-sheets structure of Aβ fibrils by interacting between the backbone C=O group from one strand and the N–H group from the other strand of Aβ fibril (Krebs et al. 2005); (Fig. 1). Thio-S also can bind with lipid membranes or lipid compounds in the cell, but produces high background when observed under a fluorescent microscope (Liu et al. 2008), so, Thio-S can be used for qualitative measurement of plaques, but the actual quantity cannot be accurately determined. As such, Thio-S does not provide an accurate

Fig. 8 Morphological characterization of Aβ plaques with differ-ent amyloid dyes. After labeling of 5×FAD sections with Cur, NC, Thio-S, CR, and 6E10, the Aβ plaques were categorized into differ-ent types. Both Cur and NC showed four distinct types of Aβ plaques, such as core, neurite, diffuse, and burned-out, similar to those plaques revealed by Thio-S staining. The diffuse plaques labeled by Thio-S were not as distinct as those labeled for Cur or NC, and also were

very uncommon (1 plaque/5–10 microscopic fields, ×20 objective). Use of CR failed to label these morphologically diverse plaques, especially neuritic plaques and diffuse plaques. The Aβ-specific anti-body (6E10) labeled three of these types of plaques distinctly, but it did not reveal neuritic plaques as distinctly as those observed with Cur-, NC-, or Thio-S-stained sections. Scale bar indicates 25 μm and is applicable to all other images

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measure of Aβ plaque numbers, especially when anionic phospholipids are present in the tissue (Bussiere et al. 2004), which can lead to inflated counts of Aβ plaques. Consequently, the high background/artifact with Thio-S is less than optimal for labeling Aβ fibrils (Hudson et al. 2009). Moreover, Thio-S cannot be used to study Cur-treated animal tissue, because both Cur and Thio-S have similar excitation/emission (430/550 nm) wave lengths (Hudson et al. 2009), so, fluorescent properties of Cur over-lap with the wavelength of Thio-S fluorescence, producing high background levels in cells/tissue.

Similarly, a classical amyloid-binding dye, Congo red (CR), has been used to identify the Aβ plaques in brain tis-sue (Elghetany and Saleem 1988; Elghetany et al. 1989)

under alkaline conditions in recent years. In the presence of alkaline solution, along with high salt concentrations, CR binds with Aβ plaques and produces an apple-green bire-fringence (Fig. 10, lower panel) which can be observed by using polarized light (Elghetany and Saleem 1988; Elghet-any et al. 1989). It is thought that CR intercalates between the β-strands, parallel to the peptide chains and perpen-dicular to the fibril direction (Klunk et al. 1989); (Fig. 1). The negatively charged sulfonate group of CR binds with the positive N-terminals of Aβ polypeptides to produce this birefringence (Cooper 1974; Wu et al. 2007). However, in the present study, we did not use alkaline variant of CR, but we have used 1 % NaOH solution to obtain an optimal alkaline condition so that CR could bind with Aβ plaques

Fig. 9 Labeling of Aβ plaques with different concentrations of Cur and NC. Sections from 5×FAD mice were stained with Cur or NC at concentrations ranging from 1 mM to1 nM. Fluorescent intensity (arbitrary unit: AU) of Aβ plaques stained by different concentrations of Cur (Graph A) and NC (Graph B) after subtracting the background fluorescent signal from respective wild-type tissue stained with Cur

or NC. Note: both Cur and NC labeled Aβ plaques, even in 1 nM con-centration, but minimum concentrations of 10 nM were required to observe clear images of plaques. The scale bar indicates 100 μm and is applicable to all other images. **p < 0.01 compared to 1 mM, 100, 10 and 1 μM treated groups (Graph A), whereas ##p < 0.01 compared to 1 mM, 100, 10, 1 μM, 100 and 10 nM treated groups (Graph B)

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effectively. With this method, we were able to observe high signal-to-noise ratio (SNR) when labeling Aβ. Of course, if we had used alkaline variant of CR, it may have provided a better SNR for Aβ labeling, which might make it more comparable with Cur or NC, Thio-S, and 6E10. However, in the present study when we labeled Aβ plaques with dif-ferent concentrations of CR (1, 0.5, 0.25, and 0.125 %), we observed that at the concentrations of 1, 0.5, and 0.25 % the labeling of Aβ plaques was not different (Fig. 10), whereas 0.125 % labeled significantly less Aβ plaques compared to other three groups (Elghetany et al. 1989; Wu et al. 2012), suggesting that get optimum Aβ-plaque labeling requires at least 0.25 % of CR solution.

The main objective of this study was to focus on how dietary Cur and NC can be used to label Aβ plaques from 5×FAD brain tissue. In terms of specificity of binding, when we stained wild-type tissue with Cur or NC, we observed a very faint green fluorescent background signal, whereas in 5×FAD tissue we observed a robust green fluo-rescent signal, coming exclusively from Cur and NC when bound to Aβ plaques. To show the specificity of Cur bind-ing to Aβ plaques and oligomers, we used 6E10 (which can bind specifically with 1–16 residues of N-terminal site of most Aβ species, including Aβ-monomers, oligomers, and fibrils/plaques) and A11 (Aβ-oligomer-specific antibody). Surprisingly, we observed that both Cur and NC were

Fig. 10 Labeling of Aβ plaques with different concentration of Congo red solution. 5×FAD sections were stained with different con-centrations of Congro red solutions (1, 0.5, 0.25 and 0.125 %) dis-solved in 80 % ethanol, along with 1 % NaOH solution, and images were taken either using a compound light microscope (upper panel) or polarized microscope (birefringence, lower panel). A number of

Aβ plaques were counted using imageJ. Note: statistically equivalent number of Aβ plaques were observed (graph) at the dose of 1, 0.5, and 0.25 % of CR solution, whereas 0.125 % of CR solution labeled significantly fewer Aβ plaques in comparison with those labeled by the other three doses. Scale bar indicates 250.5 μm and is applicable to all other images. **p < 0.01 compared to 1, 0, and 0.25 % groups

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co-localized almost completely with different species of Aβ, which suggests that Cur and NC are very specific to Aβ oligomers (Fig. 4) and fibrils (Fig. 3) (Ono et al. 2004; Garcia-Alloza et al. 2007; Mutsuga et al. 2012; Tei et al. 2012). In addition to binding of extracellular Aβ plaques, Cur and NC labeled intracellular Aβ aggregates and co-localized with Aβ antibody (Fig. 5), suggesting that Cur and NC can label not only extracellular Aβ plaques, but also Aβ deposited in intracellular spaces, re-confirming its high affinity toward Aβ.

However, when we compared the number of plaques labeled by Cur and NC with other classical amyloid dyes in tissues of the cortex (Fig. 7b) and dentate gyrus of the hippocampus in mice (Fig. 7c), we found that the num-ber of Aβ plaques labeled by CR and Thio-S were signifi-cantly less than those detected by dietary Cur, NC, and Aβ antibody, suggesting that these classical markers have less affinity for binding to Aβ plaques than Cur or NC. Although we have carefully counted the number of plaques on single focal plane, future work, using unbiased stereol-ogy, is needed to assess plaque load in regions of interest to obtain a more comprehensive profile of labeling capacity of Cur and NC.

Morphological characterization of different Aβ plaques is important in AD research (Bussiere et al. 2004; Serrano-Pozo et al. 2011). On the basis of morphological charac-teristics, researchers have divided the Aβ plaques into four categories: core, neurite, diffuse, and burned-out (Bussiere et al. 2004). Categorization of different Aβ plaques provides insights into the evolution of plaques and their loads, espe-cially after treatment with any neuroprotective drugs (Ser-rano-Pozo et al. 2011). For example, diffuse Aβ plaques are commonly present in the brains of cognitively intact elderly people, whereas dense-core plaques, particularly those with neuritic dystrophies, are most often found in patients with AD dementia (Serrano-Pozo et al. 2011). In addition, core and neuritic plaques are denser and are relatively larger than diffuse or burned-out plaques (Fig. 8). Anti-amyloid drug treatments may decrease the amount of core and neu-rite plaques and may convert them into diffuse or burned-out plaques (Serrano-Pozo et al. 2011). However, we have observed that dietary Cur, NC, and Thio-S-stained sections labeled four distinct types of plaques, though the diffuse plaques (mesh of fibrils), stained by Thio-S, were relatively uncommon (1 plaque/5–10 microscopic fields, 20× objec-tive). In contrast, CR did not show distinctively for this diverse plaque morphology, especially for the neuritic (a central dense core surrounded by either a corona of fibril-like material), and diffuse plaques. Interestingly, Aβ anti-body (6E10) did not distinguish neuritic plaques with a dense core surrounded by a thin layer of Aβ peptide, while Cur- and NC-labeled sections clearly revealed this mor-phology (Fig. 8). Therefore, labeling of different Aβ-plaque

types may be optimized with Cur, rather than other amy-loid-binding markers or Aβ-antibody stains, given that use of Cur allows the visualization of distinct morphological characteristics.

To date, there are several well-characterized dyes, compounds, and antibodies available and, undoubtedly, they are very specific to targeted Aβ species for detect-ing Aβ plaques, but these are much more expensive than dietary Cur or NC. Moreover, detection of Aβ plaques using antibody is time consuming, taking at least 1–2 days via immunocytochemistry. It also requires several acces-sory chemicals, which is also another economic burden to researchers. On the other hand, detection of Aβ plaques by Cur or NC is very simple and rapid, requiring less than half an hour, with no need for any extra chemicals, thus becoming relatively inexpensive method. Moreo-ver, fluorescent activity of Cur/NC is very stable, with no extra care needed, requiring minimal amounts (nM level) to label Aβ plaques, and is very specific to different spe-cies of Aβ, including oligomers and fibrils, as is the case with Aβ antibodies (Table 1). Moreover, Cur is less costly, easily available, and can produce high fluorescent inten-sity when it binds to Aβ plaques. However, for detection of Aβ plaques after labeling with Cur or NC and Thio-s require a fluorescent microscope. Similarly, for detection of optimal Aβ signal (birefringence) after CR staining, polar-ized filters attached to the microscope are also required (though compound light microscope can also be used for this purpose). Although the cost may be similar when using Cur or CR for labeling and imaging of Aβ, Cur can bind and label most of the Aβ species (Figs. 3, 4) (Yang et al. 2005), whereas CR binds protofibrils and fibrils only (Wu et al. 2007; Xu et al. 2014). Therefore, Aβ labeling can be achieved more efficiently by Cur or NC than by CR, for the same cost (Table 1).

In addition to its use for determining the minimum dose required for Cur and NC to label Aβ plaques in brain tissue, 5× FAD brain sections were stained with different doses of Cur and NC, producing noticeable labeling of Aβ plaques that could be observed, even at 1 nM concentration of Cur or NC (Fig. 9). This concentration (1 nM) does not provide the optimal fluorescent intensity to count the number of plaques, but the 10 nM concentration of both dietary Cur and NC does. When measuring fluorescent intensity of plaques stained by both Cur and NC, we observed that NC had greater fluorescent signals upon binding to Aβ plaques, in all these concentrations, compared with Cur. This may be due to the fact that the NC is made up in solid lipid par-ticles, which are hydrophobic in nature, allowing it to pen-etrate into the hydrophobic core of the Aβ plaques (Soto et al. 1994a), which may account for the increased fluo-rescent intensity of Aβ plaques, relative to dietary Cur. We also suspect that, because of its hydrophobic or lipophilic

Histochem Cell Biol

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Tabl

e 1

Com

pari

sons

of

use

of C

ur, N

C, T

hio-

S, a

nd C

R f

or la

belin

g an

d im

agin

g Aβ

pla

ques

rel

ativ

e to

Aβ

-spe

cific

ant

ibod

y

Feat

ures

Aβ

-ant

ibod

yC

ur a

nd N

CT

hio-

SC

ongo

red

Dur

atio

n of

sta

inin

gN

eed

~24

to 4

8 h

for

imm

unoh

isto

chem

-ic

ally

loca

lizat

ion

of Aβ

pla

ques

10–3

0 m

in10

min

60 m

in

Acc

esso

ry c

hem

ical

sR

equi

red

seve

ral a

cces

sory

che

mic

als,

in

clud

ing

seco

ndar

y an

tibod

ies

for

dete

ctio

n of

Aβ

pla

ques

in ti

ssue

Not

hing

is r

equi

red

othe

r th

an C

ur/N

C

and

met

hano

l for

Aβ

-pla

que

dete

ctio

n in

pos

tmor

tem

bra

in ti

ssue

Req

uire

s a

few

che

mic

als,

suc

h as

et

hano

lR

equi

res

very

few

che

mic

als

such

as

NaO

H, e

than

ol

Cos

tC

ostly

: one

Aβ

-spe

cific

ant

ibod

y vi

al

requ

ire

~$20

0 to

300

(U

SD)

Cos

t eff

ectiv

e: 1

g C

ur c

ost ~

$5 (

USD

) an

d ca

n be

use

d or

sev

eral

oth

er ty

pes

of ti

ssue

Cos

t eff

ectiv

e: 1

g ~

$5 (

USD

) ca

n be

us

ed f

or f

ew ti

ssue

sC

ost e

ffec

tive:

1 g

CR

cos

t >$5

(U

SD)

and

can

be u

sed

for

seve

ral t

issu

es

Spec

ifici

tyD

iffe

rent

ant

ibod

ies

are

requ

ired

for

Aβ

ol

igom

ers

and

fibri

lsC

ur a

nd N

C c

an b

ind

to b

oth

Aβ

olig

om-

ers

and

fibri

lsC

an b

ind

only

fibr

ils, n

ot m

onom

ers

or

olig

omer

sC

an o

nly

bind

with

Aβ

pro

tofib

rils

and

fib

rils

(W

u et

al.

2007

, 201

2)

Stab

ility

Dep

ends

on

the

dye

atta

ched

to s

econ

d-ar

y an

tibod

yV

ery

stab

le, e

ven

in r

oom

tem

pera

ture

w

hen

boun

d w

ith Aβ

Stab

le in

met

hano

lSt

able

in e

than

ol

Car

e af

ter

stai

ning

Nee

ds e

xtra

car

e af

ter

stai

ning

, suc

h as

be

ing

kept

in th

e da

rk a

nd f

roze

n al

l th

e tim

e

Not

as

light

-sen

sitiv

e an

d m

ore

stab

le a

t ro

om te

mpe

ratu

reL

ight

sen

sitiv

eN

ot li

ght s

ensi

tive

Mic

rosc

ope

requ

ired

Com

poun

d lig

ht o

r flu

ores

cent

(de

pend

-in

g on

use

of

seco

ndar

y an

tibod

y)Fl

uore

scen

tFl

uore

scen

tL

ight

mic

rosc

ope

or p

olar

ized

mic

ro-

scop

e or

pol

ariz

e fil

ter

Bac

kgro

und

stai

ning

Gen

eral

ly n

o ba

ckgr

ound

Ver

y le

ss b

ackg

roun

dH

igh

back

grou

nd d

ue to

bin

ding

with

lip

id m

embr

ane

or li

pid

com

poun

ds

pres

ent i

n ce

ll

Les

s ba

ckgr

ound

In v

ivo

Aβ

imag

ing

May

not

be

appl

icab

leH

ighl

y ap

plic

able

May

not

be

appl

icab

leM

ay n

ot b

e ap

plic

able

Histochem Cell Biol

1 3

nature, NC might bind with C-terminal region of Aβ, which is basically hydrophobic (Soto et al. 1994b). Therefore, we suggest that 10 nM may be the minimum concentration for NC and 1 μM for Cur required for proper labeling and imaging of Aβ plaques (Fig. 9a and b). Recently, Ran and colleagues (2009) reported that even 0.2 nM Cur can bind with Aβ in vitro, but whether this dose is sufficient to label Aβ plaques in tissue is still not clear.

Although the present study was focused on labeling post-fixed brain tissue from 5×FAD to identify Aβ plaques, it can also be used to label in vivo Aβ-plaque load, as reported by Lazar and colleagues (2013), using an advanced imag-ing microscope. However, to show whether Cur and NC can cross blood brain barrier and label Aβ plaques in vivo, we also injected Cur and NC for 2 and 5 days, intraperitoneally, and observed that they both labeled Aβ plaques, to a simi-lar extent as observed in postmortem tissue staining (Fig. 6). Because we do not know how much Cur or NC gets into the brain, further investigations with different modes of Cur and NC administration are needed. Clearly, NC produces more labeling than Cur, which might be due to its enhanced permeability into brain tissue, because it is formed from solid lipid particles (Fig. 6a). Using Aβ-specific antibody, we confirmed that Cur or NC only bound with Aβ plaques (Fig. 6b). Therefore, this observation suggests that Cur, especially NC, can be used for monitoring Aβ plaques in vivo and would be a potent fluorochrome for noninva-sive imaging of Aβ in studies assessing potential treatments (Garcia-Alloza et al. 2007; Lazar et al. 2013). Several Cur derivatives (structural modifications) are available, which can be applied for this purpose, as reported by several inves-tigators (Ran et al. 2009; Koronyo-Hamaoui et al. 2011; Koronyo et al. 2012; Tei et al. 2012; Lazar et al. 2013; Zhang et al. 2014). Similarly, retinal scans can also be used to monitor the deposition of Aβ plaques for screening AD patients after Cur therapy (McClure et al. 2015; Zhang et al. 2015a, b), but such developments need further research to adequately explore the exact dose and duration of Cur administration for these expanded uses.

Conclusion

As a potent, anti-amyloid polyphenol, and due to the struc-tural similarities with classical amyloid-binding dyes, Cur possesses the requisite profile for labeling and imaging of Aβ plaques in postmortem brain tissue and in vivo. Dietary Cur and NC can bind with Aβ plaques at low concentra-tions, in a very specific manner, with similar affinity to the Aβ antibody, yet show more binding capabilities than traditional amyloid-binding dyes. Taken together, our data suggest that Cur can be used as an alternative to label the Aβ plaques in postmortem brain tissue and can be used as a

quick and easy method to detect Aβ-plaque load, after anti-amyloid therapy in experimental models of AD.

Acknowledgments Support for this study came from the Field Neu-rosciences Institute, at St. Mary’s of Michigan. We thank Verdure Sci-ence (Noblesville, IN) for donating the nanocurcumin for this study.

Author contributions P.M. designed, performed the experiments, and collected the data. T.H., N.K., L.P. and C.L. helped with tissue processing. P.M., G.L.D., and J.R. analyzed the data and wrote the manuscript.

Compliance with ethical standards

Conflict of interest Authors declare that there is no conflict of interest to publish this research article.

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