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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: J Neuroimmune Pharmacol. 2020 Sep 5;16(3):592–608. doi: 10.1007/s11481-020-09955-2

Reduction of Lewy body pathology by oral cinnamon

Sumita Raha 1, Debashis Dutta 1, Avik Roy 1, Kalipada Pahan 1,2
PMCID: PMC7933354  NIHMSID: NIHMS1626617  PMID: 32889602

Abstract

α-Synucleinopathies in a broader sense comprise of several neurodegenerative disorders that primarily include Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These disorders are well characterized by the accumulation of aggregated insoluble α-synuclein (α-syn) protein known as Lewy bodies. Till date no effective cure is available to reduce the burden of Lewy body. The present investigation underlines the importance of a naturally used spice and flavoring agent viz. cinnamon in reducing α-syn deposits in transgenic mice expressing mutant A53T human α-syn. Upon oral administration, cinnamon markedly reduced the level of insoluble α-syn in nigra, hippocampus and brain stem of A53T mice. We also demonstrated that sodium benzoate (NaB), a metabolite of cinnamon, a widely used food additive and a FDA-approved drug for glycine encephalopathy, was also capable of reducing α-syn deposits in A53T mice. In addition, both cinnamon and NaB treatments showed improvement in their motor and cognitive functions. Glial activation plays an important role in the pathogenesis of various neurodegenerative disorders including PD, DLB and MSA, and we found suppression of microglial and astroglial activation in the nigra of A53T mice upon cinnamon treatment. Moreover, neuroprotective proteins like DJ-1 and Parkin are known to reduce the formation of Lewy bodies in the CNS. Accordingly, we observed upregulation and/or normalization of DJ-1 and Parkin in the nigra of A53T mice by treatment with cinnamon and NaB. Together, these results highlight a new therapeutic property of cinnamon and suggest that cinnamon and NaB may be used to halt the progression of α-synucleinopathies.

Keywords: α-synucleinopathy, A53T mice, Cinnamon, Sodium benzoate, Glial activation

Introduction

α-Synuclein (α-syn) is the key molecule in the pathogenesis of synucleinopathy that leads to neurodegenerative diseases characterized by the abnormal accumulation of α-syn aggregates in neurons, nerve fibers or glial cells of which the main types are Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Of these, PD is the second most devastating progressive neurodegenerative movement disorder caused by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and is characterized by accumulation of insoluble α-syn aggregates referred to as Lewy bodies (LB) (Luk et al., 2012). The clinical manifestations of PD patients are at least two of four cardinal features namely, bradykinesia (slowness and minimal movement), rigidity, resting tremor (trembling), and an impairment of postural balance leading to disturbance of gait and falling (Lang and Lozano, 1998; Chavez-Bejar et al., 2008; Balestrino and Schapira, 2020). It has been established that the pathological α-syn can transmit from cell-to-cell in a prion-like fashion to promote the neurodegeneration in these diseases (Spillantini et al., 1997). Despite intense research, until now, no effective therapy is available to reduce α-synucleinopathy. Therefore, finding a safer and more effective approach to inhibit the progression of PD remains an active area of research.

Plants are known to have a long history, as a therapeutic resource for drug discovery. One such plant product is cinnamon, obtained from the bark of cinnamon tree, which has already been being used for centuries throughout the world as a common food spice and flavoring agent (Pahan, 2015). Upon metabolism, cinnamon is known to be converted into sodium benzoate (NaB). Interestingly, NaB is a widely-used food-additive and a FDA-approved drug for glycine encephalopathy (Neuberger et al., 2000). Being a component of Ammonul®, it is also a FDA-approved medication for urea cycle disorders. Recent studies from our lab have indicated that cinnamon may be a helpful therapeutic for multiple sclerosis (MS) and is capable of modifying pathological features in mice with experimental allergic encephalomyelitis (EAE), an animal model of MS (Brahmachari and Pahan, 2007; Pahan, 2011; Mondal and Pahan, 2015). Cinnamon and NaB are also capable of protecting dopaminergic neurons in the nigra and restoring dopamine in the striatum in MPTP mouse model of PD (Khasnavis and Pahan, 2014; Patel et al., 2019).

The present investigation was carried out to examine whether cinnamon and NaB have any protective effect against α-syn-mediated toxicity in transgenic mice expressing mutant A53T human α-syn. Here, we provide evidence that oral feeding of ground cinnamon and NaB markedly lowered α-syn deposits from different parts of the brain of A53T mice to improve locomotor and cognitive performances. Moreover, consistent to the involvement of microglial activation and loss of neuroprotective proteins like DJ-1 and Parkin in α-synucleinopathy, we found attenuation of microglial activation and upregulation of DJ-1 and Parkin in the nigra by cinnamon treatment. These results suggest that oral cinnamon and its metabolite NaB may be one of the safest approaches to clear α-syn depositions from the brain of patients with PD, DLB and MSA.

Materials and Methods

Reagents and antibodies

Antibodies, their applications, sources and dilutions are listed in Table S1. Original Ceylon cinnamon (Cinnamonum verum) in ground form was obtained from Indus Organics (San Ramon, CA). All molecular biology-grade and chemicals were obtained from Sigma-Aldrich or Bio-Rad. IR-Dye-labeled secondary antibodies used for immunoblotting was from Li-Cor Biosciences.

Animals and cinnamon treatment

Mice were maintained and experiments conducted in accordance with National Institute of Health guidelines and were approved by the Rush University Medical Center IACUC. Mice were maintained in a 14 h light, 10 h dark cycle with continuous supply of food and water. A53T mice (Strain-B6; C3-Tg (Prnp-SNCA*A53T) 83Vle/J) were obtained from Jackson Laboratories. Mice were treated with cinnamon (100 mg/kg body wt/d) and NaB (50 mg/kg body wt/d) via gavage for 60 d. Cinnamon (Cinnamonum verum) powder and NaB were mixed in 0.5% methylcellulose (MC). Mice were gavaged 100 μL cinnamon-mixed MC and 100 μL NaB-mixed MC once daily using gavage needle as described (Jana et al., 2013; Khasnavis and Pahan, 2014; Modi et al., 2015; Patel et al., 2019).

Protein extraction

Total protein was extracted from midbrain, hippocampus and brain stem using two different extraction buffers: Insoluble buffer and RIPA buffer (Thermo Scientific, Waltham, MA). Samples were homogenized on ice, sonicated for 10 s, and centrifuged at 15,000 g for 30 min at 4°C.

Separation of soluble and insoluble protein

Brain tissues were homogenized in 5 volumes of Triton X-100 buffer (1% Triton X-100, 125 mM NaCl, 20 mM NaCl, 5 mM EDTA, 1.5 mM MgCl2, 10% glycerol) supplemented with protease and phosphatase inhibitors (Sigma) using a sonicator set at pulse 2 for 10s. The brain lysates were centrifuged at 15,000 g for 30 min at 4°C. The collected supernatant was considered the soluble fraction. The pellet was then extracted in 2.5 volumes of 2% SDS, 50 mM Tris-HCl, 125 mM NaCl, 1 mM EDTA and protease and phosphatase inhibitors, followed by sonication with similar settings. The re-suspended pellet was centrifuged at 15,000 g for 30 min at 25 °C. The supernatant was considered as the insoluble fraction. The protein concentration was determined by Bradford assay (Bio-Rad).

Immunoblotting and densitometric analyses

The SDS-soluble and insoluble fractions and whole lysates were run for western blots to evaluate levels of monomeric, soluble and oligomeric α-syn. The supernatant was aliquoted and stored at −80°C until use. Protein concentrations were determined using a NanoDrop 2000 (Thermo Fisher), and 15–30 μg sample was heat-denatured and resolved on either 12% or 15% polyacrylamide-SDS gels in MES buffer (50 mM MES, 50 mM Tris base, 0.1% SDS, 1 mM EDTA, pH 7.3) or 1X SDS Running Buffer. Proteins were transferred onto 0.45μm nitrocellulose membranes in Towbin Buffer (25 mM Tris, 192 mM glycine, 20% (w/v) methanol) under wet conditions (40V for 120 min). Membranes were blocked for 1 hr with blocking buffer (Li-Cor), incubated with primary antibodies (Table S1) overnight at 4ºC under shaking conditions, washed, incubated with IR-dye labeled secondary antibodies (1:5,000; Li-Cor) for 45 min at room temperature, washed and visualized with the Odyssey Infrared Imaging System (Li-Cor). Blots were converted to binary, analyzed using Image J software and normalized to the loading control (β-actin).

Immunohistochemistry (IHC)

For immunohistochemistry, mice were anesthetized and intracardially perfused with 1X PBS followed by 4 % paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4. The brains were post fixed in PFA overnight at 4 °C, and were then transferred to phosphate buffer containing 30% sucrose at 4 °C. Coronal brain sections were cut and saved in serial order at −20 °C until immunostained. For this the hemi brains incubated in 30% sucrose were washed thoroughly in PBS cryo-sectioned using a sliding microtome (American opticals 860). Prior to staining, 40uM free floating hippocampal sections were washed thoroughly in PBS. The sections were blocked using 2% BSA in PBSTT (PBS+Triton X-100+Tween-20) for 1h. Next, the sections were incubated with primary antibody in 1% PBSTT at 4°C overnight. The following day, sections were washed in PBSTT and incubated with 488 or 647- conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for 3 h at room temperature. Following washes in PBSTT, the sections were mounted on glass sides (Ghosh et al., 2007; Khasnavis and Pahan, 2012). The samples were visualized under OlympusBX41 fluorescence microscope equipped with a Hamamatsu ORCA-03G camera. For DAB staining, the nigral, hippocampal and brain stem sections were stained for α-syn and p-syn (p129S) using the Vectastain DAB protocol, mounted and observed under a Olympus bright field microscope. Optical Density measures were produced using ImageJ software (Ghosh et al., 2009) as described before (Varghese et al., 2014).

Gas chromatography mass spectrometric analysis for detecting NaB

After cinnamon treatment, hippocampal extracts from A53T mice were homogenized in ice-cold non-detergent hypotonic buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 100 mM DTT, protease and phosphatase inhibitor cocktail]. After 10 min of additional incubation in the hypotonic buffer, the homogenate was centrifuged at 8,000 g at 4°C for 10 min. Next, the pellet was homogenized in ice-cold extraction buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.21 M NaCl, 0.2 mM EDTA, 25% (v/v) glycerol, 100 mM DTT, protease and phosphatase inhibitor cocktail], placed on a rotating shaker at 4°C for 1 h, and then centrifuged at 18,000 g for 10 min. The eluted fraction was then transferred to methanol: chloroform: water (4:3:1) mixture and then centrifuged at 14,000 rpm for 90 sec. The organic phase was collected, evaporated in the speedvac, reconstituted with 30μL chloroform, and finally analyzed in Thermo Scientific™ ISQ™ 7000 mass spectrometry (GC-MS) system (Jana et al., 2013).

Behavioral Analyses

Open field test

It was performed as described earlier (Patel et al., 2019; Patel et al., 2020). Briefly, each mouse was allowed to freely explore an open field arena for 5 min. The testing apparatus was a classic open field (i.e., a wooden floor square arena, 40 × 40 cm, with walls 30 cm high). A video camera (Basler Gen I Cam-Basler acA 1300–60) connected to a Noldus computer system was placed above the box. Each mouse was placed individually on the center of the arena and the locomotor activity and other parameters like velocity, total distance travelled and center time frequency was monitored for 5 minutes using live video tracking system (Noldus System). The central area was arbitrarily defined as a square of 20 × 20 cm (half the total area).

Rotarod test

The motor coordination of mice was measured on a Rotarod apparatus (ENV-576M; Med-associates Inc.), using protocol described earlier (Chandra et al., 2016; Patel et al., 2019). Briefly, mice were transported (within their home cage) to acclimate to the testing room for 1 h prior to trial. Before acquisition, the parameters of the Rotarod system equipped with automatic fall detector such as start speed and acceleration were carefully checked before acquisition. Each mouse was placed on the confined section of the rod and trial was initiated with a smooth increase in speed from 4 rpm to 40 rpm for 5 mins. If the mouse did not fall from the rod, it was removed from the rod after 5 min.

Pole test

It was performed as described before (Chandra et al., 2016). A vertical wooden pole with a rough surface (50 cm in height and 1 cm in diameter) was placed in the home cage. Mice were placed head-up on top of the pole, so that they could orient themselves downward and descend the pole back into their home cage. On the test day, animals were exposed to five trials. The time spent to orient downward (t-turn) and the time to descend (t-descend) were measured.

Barnes maze

Barnes maze test was performed as for evaluating the spatial memory of the mice as described previously (Corbett et al., 2015; Roy et al., 2016; Modi et al., 2017). Mice were trained for two days on a 20-hole Barnes maze where only one tunnel contained colored food bait. Following two days of training, mice were given rest for one day and tested on the maze on the fourth day. During training, mouse was placed in the middle of the maze in a cylindrical 10cm high start chamber. After 10s, the cylinder was removed and the mouse was allowed to freely move and explore the maze in order to find the food-baited tunnel. The maze was well lit with high wattage light to generate sufficient light and heat to motivate the mouse to find and escape into the tunnel. On the day of the test, the mice were deprived of food and their performance was recorded using the Noldus system and EthoVisionXT software. Memory of the mouse was analyzed by latency to the goal box (duration before all four paws were on the escape box floor) and number of errors (incorrect responses before all four paws were on the escape box floor).

Statistical analysis

Statistical analysis was conducted, using Graph Pad Prism 7.0c software. Unless otherwise stated, one-way or two-way ANOVA followed by Dunnett’s post-hoc analyses was performed to determine the significance of differences among multiple experimental groups. Student’s t test was used when the significance of differences was determined between two groups. Data were expressed as mean ± standard error (SEM) or mean ± standard deviation (SD), and values with P<0.05 were considered statistically significant.

Results

Oral administration of cinnamon decreases α-syn and pSer129-syn in the nigra of aged A53T mice

We first evaluated the efficacy of oral administration of cinnamon in lowering α-syn aggregates in A53T mice. Ten-month-old A53T mice were oral gavaged with cinnamon (100 mg/kg/day) or vehicle (0.5% methylcellulose) for 2 months followed by immunostaining of the midbrain sections using antibodies against α-syn and TH (Fig. 1A). A remarkable increase in the levels of α-syn was observed in A53T mice when compared with non-Tg mice (Fig. 1A). However, oral administration of cinnamon markedly reduced the levels of α-syn in the nigra of A53T mice (Fig. 1A). Quantification of α-syn immunostaining indicated a significant reduction of α-syn in cinnamon-treated mice relative to the vehicle-treated group (Fig. 1B). We further validated the expression α-syn using DAB immunohistostaining of the mid brain region. The results are at par with the immunostaining images where we confirmed a significant reduction in the total α-syn levels in cinnamon treated mice when compared to the vehicle-treated group (Fig. 1E). Quantification of average optical density for DAB stained section was done using NIH ImageJ. The relative optical density of total α-syn exhibited a significant decrease in cinnamon-treated A53T mice as compared to vehicle-treated ones (Fig. 1F). Immunoblot analyses were done using both insoluble and soluble protein extracts from midbrain tissues. Levels of both soluble and insoluble α-syn was more in the nigra of A53T mice as compared to non-Tg mice (Fig. 1I–L). However, cinnamon treatment decreased the level of insoluble, but not soluble, α-syn in the nigra of A53T mice (Fig. 1I–L).

Figure 1. Oral administration of cinnamon mitigates α-syn and p-syn pathology in the nigra of aged A53T mice.

Figure 1.

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Ten months old A53T mice (n=5/group) were orally gavaged with cinnamon (100 mg/Kg/day) and vehicle (0.5% methylcellulose) for two months. Ventral midbrain sections were double-labeled with α-syn antibody (red) and TH antibody (green) (A). MFI quantification of α-syn (B). Sections were double-labeled for p-syn (red) and TH (green) (C). MFI quantification of p-syn (D). Sections were DAB-stained for α-syn (E). Higher magnification images are in bottom panel. Relative optical density of α-syn (F). Sections were DAB-stained for p-syn (G). Higher magnification images are in bottom panel. Relative optical density of p-syn (H). A total of nine images (20X magnification) from 5 different mice per group were quantified. Representative immunoblot showing detection of soluble α-syn in nigral homogenates (I). Densitometric quantification of α-syn normalized to β-actin (J). Representative immunoblot showing detection of insoluble α-syn levels in nigral homogenates (K). Densitometric quantification of monomeric α-syn normalized to β-actin (L). Data are mean ±SEM for 4 mice per group. All statistical analysis were performed by student’s t test and one way ANOVA followed by Dunnett’s multiple comparison test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Several post-translational modifications of α-syn are known to occur in PD, DLB and MSA. Among them is phosphorylation at Ser129 [phosphorylated α-synuclein (p129S)](p-syn), a modification that may be critical for disease pathogenesis. Phosphorylation appears to play an important role in fibrillogenesis, LB formation, and neurotoxicity of α-syn in vivo. We therefore evaluated whether oral feeding of cinnamon was effective in reducing p-syn pathology in A53T mice. Co-labeling of nigral sections with p-syn and TH showed significantly higher level of p-syn deposition in A53T mice when compared to non-Tg mice (Fig. 1C). Similar results were observed with DAB immunostaining (Fig. 1G). However, cinnamon treatment significantly decreased the level of p-syn in A53T mice as compared to vehicle treatment (Fig. 1C, G). Quantitative analysis of p-syn expression also confirmed a significant decrease of p-syn levels in the nigra of cinnamon-fed A53T mice as compare to vehicle-fed ones (Fig. 1D, H).

Cinnamon treated aged A53T mice shows a mitigation of Lewy body pathology in the hippocampus

The onset of Lewy body pathology and synuclenopathies in the hippocampus leads to dementia, a non-motor function of DLB and some PD patients. In order to determine the total α-syn accumulation and distribution, DAB staining was done on free-floating hippocampal sections. A significant increase in the levels of α-syn was observed in the A53T mice when compared to the non-Tg mice (Fig. 2A, B). However, oral administration of cinnamon significantly reduced the levels of total α-syn in the hippocampus of cinnamon-treated A53T mice (Fig. 2A, B). We further evaluated the CA1 region of the hippocampus with immunofluorescence studies using p-syn and neuronal marker NeuN. Our results indicate marked increase in p-syn expression throughout the CA1 region of the hippocampus of A53T mice as compared to non-Tg mice (Fig. 2C, D). However, cinnamon-fed A53T mice showed substantial reduction in p‐syn levels as compared to vehicle-treated mice (Fig. 2C, D). The expression of neuronal marker was not significantly different between control and cinnamon-treated mice. The level of p-syn was also analyzed using ImageJ (Fig. 2D). Similar results were further validated with DAB staining using p129S antibody (Fig. 2E, F). Immunoblot analysis was performed from hippocampal homogenates of all groups and probed with α-syn-specific antibody. Our results indicate a significant increase in α-syn expression in the hippocampus of A53T mice as compared to non-Tg mice (Fig. 2G). However, cinnamon treatment showed remarkable decrease in α-syn levels in both monomeric (Fig. 2G, H) and oligomeric form (Fig. 2G, I). Densitometric quantification of α-syn immunoblots also exhibited lower levels of this protein in the hippocampus of cinnamon-fed A53T mice than vehicle-fed mice (Fig. 2H, I).

Figure 2. Oral cinnamon reduces α-synucleinopathy in the hippocampus of aged A53T mice.

Figure 2.

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Ten months old A53T mice (n=5/group) were orally administered with Cinnamon (100 mg/Kg/day) and vehicle (0.5% methylcellulose) for two months. Representative images of hippocampal sections from all groups showing immunohistochemical DAB staining for total α-syn (A). Relative optical density of total α-syn (B) A total of nine images (20X magnification) from 5 different mice per group were quantified. Higher magnification (60X) images in adjacent panel (Scale bar-5 um). Representative images of hippocampal sections showing CA1 region from all groups co immunostained with p-syn (red) and NeuN (green). (C) Quantification showing MFI of p-syn. (D) Immunohistochemical DAB staining of p-syn from the CA1 region of the Hippocampus (E). Relative optical density of p-syn (F). A total of nine images (20X magnification) from 5 different mice per group were quantified. Higher magnification (60X) images in adjacent panel (Scale bar-5um). Hippocampal protein homogenates subjected to western blots for protein expression of total α-syn showing the oligomeric and monomeric forms (G) Densitometric quantification of α-syn normalized to β-actin (H-I). All statistical analysis were performed by student’s t test and one way ANOVA followed by Dunnett’s multiple comparison test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Oral treatment with cinnamon reduces the burden of α-syn and p129S in brain stem of aged A53T mice

In addition to nigra and hippocampus, widespread α-syn pathology is seen in brain stem as well. Therefore, we investigated the effect of oral cinnamon on α-syn burden in the brain stem. A significant increase in the levels of α-syn was observed in the A53T-vehicle mice when compared to age-matched non-Tg mice. Similar to that observed in nigra and hippocampus, oral cinnamon significantly reduced the level of α-syn in the brain stem of A53T mice (Fig. 3A, B). This was also confirmed by Western blot (Fig. 3C) and densitometric quantification showing significant decrease in both monomeric (Fig. 3D) and oligomeric (Fig. 3E) forms of α-syn in brain stem of A53T mice by cinnamon treatment. Next, we examined the status of p-syn by double-labeling of NeuN and p-syn and found marked abundance of p-syn in A53T mice when compared with age-matched non-Tg mice (Fig. 3F, H). On the other hand, cinnamon treatment drastically reduced the burden of p-syn (Fig. 3F, H). These results were also confirmed by DAB staining of brain stem sections with p129S antibody (Fig. 3G, I). Together, these data indicate decrease in α-syn burden in brain stem of A53T mice by oral cinnamon.

Figure 3. Oral treatment of cinnamon attenuates α-synucleinopathy in the brain stem of aged A53T mice.

Figure 3.

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Ten months old A53T mice (n=5/group) were treated with cinnamon (100 mg/Kg/day) and vehicle (0.5% methylcellulose) orally for two months. Representative images of brain stem sections from all groups showing DAB staining for α-syn (A). Higher magnification images are in bottom panel. Relative optical density of α-syn (B). A total of nine images (20X magnification) from 5 different mice per group were quantified. Brain stem homogenates subjected to western blots for α-syn (C). Densitometric quantification of monomeric (D) and oligomeric (E) α-syn normalized to β-actin. Data are mean ±SEM for 4 mice per group. Brain stem sections were double-labeled for p-syn (red) and NeuN (green) (F). Sections were DAB stained for p-syn (G). Quantification showing MFI of p-syn (H). Relative optical density of p-syn (I). A total of nine images from 5 different mice per group were quantified. All statistical analysis were performed by student’s t test and one way ANOVA followed by Dunnett’s multiple comparison test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Cinnamon functionally alleviates motor deficits and improves cognitive functions in aged A53T mouse

We first evaluated the effect of cinnamon treatment on locomotor coordination and balance in all groups using the rotarod. As compared to vehicle-treated A53T mice, cinnamon-treated A53T mice demonstrated improved performance on the rotarod (Fig. 4A). This is in consistence to our previous reports (Khasnavis and Pahan, 2014; Patel et al., 2019) where cinnamon treatment improved MPTP-induced hypolocomotion. The pole test assesses the agility of animals and may be a measure of bradykinesia (Rial et al., 2014). The downward facing vertical pole test also revealed that, NTg mice took less time to descend the vertical pole, whereas A53T mice consistently took longer to achieve this task and displayed a significantly worse performance (Fig. 4B, C). In comparison, treatment with cinnamon significantly shortened the time that A53T mice took to make a T turn (Fig. 4B) and descend the vertical pole (Fig. 4C), suggesting an improvement in agility and bradykinesia with cinnamon treatment. We also tracked their general locomotor activity using the NOLDUS tracking software (Fig. 4D) and checked the center time frequency (Fig. 4E) in the open field arena to find significant increase in the center time frequency of cinnamon-fed A53T mice as compared to vehicle-treated mice (Fig. 4D, E).

Figure 4. Oral administration of cinnamon improves motor functions and memory in aged A53T mice.

Figure 4.

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After two months of cinnamon treatment behavioral tests (Rotarod, Pole test and Barnes maze) were performed for evaluating the motor and cognitive performances of A53T mice (n=5 or 6/group). Rotarod test was done to demonstrate feet movement of mice on a rotating rod and the latency of each mouse to fall on the base (A). Pole test evaluating the ability of the mice to grasp and maneuver on a pole (B). Time taken to take a turn in order to descend the pole (C). Open field test showing representative heat map demonstrating general locomotor activity (D) and center time frequency (E). Barnes maze test showing representative heat maps (F), latency to the goal box (G), total number of errors made (H). All data are represented as mean ±SEM of 5 or 6 mice per group. One way ANOVA followed by Dunnett’s multiple comparison test was used for statistical analysis; * p < 0.05; ** p < 0.01; *** p <0.001.

Since hippocampus of aged A53T mice is rich in aggregated α-syn and p-syn, we also monitored spatial learning and memory by Barnes maze and observed memory impairment in aged A53T mice as compared to age-matched NTg mice (Fig. 4F). As supported by the heat map (Fig. 4F), our data showed that cinnamon-treated A53T mice performed better as they took significantly less time to reach the goal box (latency) (Fig. 4G) with fewer errors (Fig. 4H) compared to the vehicle-treated mice, indicating improvement in memory and learning of A53T mice by oral cinnamon. Overall, our results suggest that oral administration of cinnamon is capable of increasing motor functions and decreasing memory deficits in aged A53T mice.

Cinnamon treatment attenuates glial activation and neuroinflammation in aged A53T mice

Neuroinflammation is one of the neuropathological features of PD (Tansey and Goldberg, 2010) and most animal models of α-synucleinopathy, including A53T mice. Moreover, microglial activation is intimately coupled to α-synucleinopathy in which aggregated α-syn leads to microglial activation (Sarkar et al., 2020) and conversely, microglial activation also potentiates α-synucleinopathy (Choi et al., 2020b; La Vitola et al., 2020). Therefore, we investigated whether oral administration of cinnamon powder was capable of reducing glial inflammation. Microglia are innate immune cells residing in the CNS and Iba1 is a prototype microglial marker (Imai and Kohsaka, 2002). Untreated A53T mice when compared to non-Tg mice exhibited marked microglial activation in the nigra with increased Iba1 expression that also co-expressed iNOS (Fig. 5A). On the contrary, there was a significant decrease in nigral Iba1-positive as well as (Iba1+iNOS)-positive microglia (Fig. 5A). This was also confirmed by counting of Iba1-positive and iNOS-positive cells (Fig 5C, E). Although microglia are more inflammatory than astrocytes, the major cell type in the CNS are astrocytes, hence, astroglial activation is also an important pathological event in neurodegenerative disorders. To determine alterations in the activated state of astrocytes, sections were double-labeled with glial fibrillary acidic protein (GFAP), an astrocytic marker, and iNOS (Fig. 5B). Similar to the suppression of microglial activation, cinnamon treatment also decreased astroglial activation as evident from decreased number of GFAP-positive and iNOS-positive cells in the nigra of aged A53T mice (Fig. 5B, D, E). Our western blot results also exhibited a significant increase in iNOS, Iba1 and GFAP in nigral tissue lysates of aged A53T mice when compared to non-Tg ones (Fig. 5F–I). However, oral administration of cinnamon significantly reduced the levels of iNOS, Iba1 and GFAP (Fig. 5F–I). Therefore, oral cinnamon is capable of reducing microglial and astroglial inflammation in the nigra of aged A53T mice.

Figure 5. Cinnamon treatment attenuates glial activation and neuroinflammation in A53T mice.

Figure 5.

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Ten months old A53T mice (n=5/group) were orally administered with cinnamon (100 mg/Kg/day) and vehicle (0.5% methylcellulose) for two months followed by double-label immunofluorescence of nigral sections for Iba1 & iNOS (A) and GFAP & iNOS (B). Iba-positive (C), GFAP-positive (D) and iNOS-positive (E) were counted in three different sections of each of 5 mice per group using the Image J software. Next, nigral homogenates were subjected to immunoblot analysis using Iba1, GFAP and iNOS antibodies with actin as loading control (F). Densitometric quantification of IBA1 (Iba1/Actin) (G), GFAP (GFAP/Actin) (H) and iNOS (iNOS/Actin) (I). All data represents mean ± SEM of 4 mice per group. Statistical analysis were performed by one way ANOVA followed by Dunnett’s multiple comparison test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Oral administration of cinnamon produces sodium benzoate (NaB) in the hippocampus of A53T mice

Interestingly, NaB, an FDA-approved drug for urea cycle disorders and glycine encephalopathy, is produced in the body from cinnamon metabolism (Jana et al., 2013; Pahan and Pahan, 2015). Therefore, with the help of GC-MS, we examined if oral administration of cinnamon increased the level of NaB in the brain of A53T mice. For NaB standard, we detected a sharp peak representing pure NaB (Fig. S1A). While the hippocampus of vehicle-treated A53T mice did not show any peak for NaB (Fig. S1B), oral cinnamon (100 mg/kg body wt/d) treatment via gavage markedly increased the level of NaB in the hippocampus (Fig. S1C). These results demonstrate that cinnamon is metabolized into NaB and that cinnamon-derived NaB is capable of entering into the CNS of aged A53T mice.

NaB feeding decreases α-syn and p-syn in the CNS of aged A53T mice

We further evaluated the effectiveness of NaB administration in lowering α-syn and p-syn aggregates in aged A53T mice. Ten-month-old A53T mice were oral gavaged with NaB (50 mg/kg/day) for 2 months. As expected, DAB immunostaining of nigral sections showed substantial increase in the levels of α-syn in A53T mice when compared to the non-Tg mice (Fig. 6A). However, oral administration of NaB significantly reduced the levels of α-syn in the nigral region of A53T mice when compared to the vehicle (Fig. 6A). This was also confirmed by measuring relative optical density of α-syn (Fig. 6B). Next, Western blot analyses were done using both insoluble and soluble protein extracts from midbrain tissues and we found increase in both soluble (Fig. 6C) and insoluble α-syn (Fig. 6D). However, similar to cinnamon treatment, oral NaB also inhibited the level of insoluble α-syn (Fig. 6D, F) without affecting the soluble form (Fig. 6C, E). Next, we monitored the status of p-syn using DAB staining of nigral sections and found robust increase in p-syn in the nigra of A53T mice as compared to non-Tg mice, which was inhibited by NaB treatment (Fig. 6G). Quantitative assessments of relative optical density further confirmed decrease in p-syn in nigra by NaB treatment (Fig. 6H).

Figure 6. Oral administration of NaB reduces α-syn and p-syn pathology in the nigra of aged A53T mice.

Figure 6.

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Ten months old A53T mice (n=5/group) were orally gavaged with NaB (50 mg/Kg/day) for two months. Representative images showing immunohistochemical DAB staining of total α-syn from SNpc (A). Relative optical density of total α-syn (B). A total of nine images from 5 different mice per group were quantified. Magnified images are presented in adjacent panels. Representative immunoblots showing detection of α-syn in soluble (C) and insoluble (D) fractions. Actin (bottom panel) was run as loading control. Bands were scanned and presented as relative to actin (E, soluble α-syn; F, insoluble α-syn). Results are mean + SEM of 4 mice per group. Representative images showing DAB staining of p-syn from SNpc (G). Relative optical density of total p-syn (H). A total of nine images from 5 different mice per group were quantified. Magnified images are presented in adjacent panel. * p < 0.05; ** p < 0.01; *** p < 0.001.

In addition to nigra, α-syn aggregation is also seen in other parts of the brain (e.g. hippocampus and brain stem) and therefore, we evaluated the effect of NaB treatment on Lewy body pathology in the hippocampus and brain stem of A53T mice. As presented above, obvious increase in the levels of α-syn (Fig.7A, E) and p-syn (Fig.7B, F) was observed in hippocampus (Fig. 7A, B) and brain stem (Fig. 7E, F) of A53T mice as compared to non-Tg mice. Similar to that seen in nigra, oral NaB markedly reduced the expression of total α-syn (Fig.7A, E) and p-syn (Fig.7B, F) in hippocampus (Fig. 7A, B) and brain stem (Fig. 7E, F) of A53T mice. Quantification of relative optical density further confirmed these results (Fig. 7C, D, G, H).

Figure 7. Oral administration of NaB mitigates α-synucleinopathy in hippocampus and brain stem of aged A53T mice.

Figure 7.

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Ten months old A53T mice (n=5/group) were orally gavaged with NaB (50 mg/Kg/day) for two months. Representative images of hippocampal coronal sections showing immunohistochemical DAB staining for total α-syn (A) and p129S (B). Relative optical density of total α-syn and p-syn (C, D). A total of nine images from 5 different mice per group were quantified. Representative images of brain stem sections showing DAB staining for total α-syn (E) and p-syn (F). Relative optical density of total α-syn and p-syn (G, H). A total of nine images (20X magnification) from 5 different mice per group were quantified. *** p < 0.001.

NaB improves behavioral deficit in A53T mice

Since NaB treatment decreased α-synucleinopathy in the CNS of A53T mice, we also monitored locomotor and cognitive performances. Similar to cinnamon treatment, NaB-treated A53T mice also exhibited improved locomotor performance in rotarod test (Fig. 8A) as well as pole test (Fig. 8B, C). Next, we monitored spatial learning and memory in NaB-treated A53T mice by Barnes maze. Our data showed that NaB treated A53T mice performed better as they took significantly less time to reach the goal box (latency) and made less errors compared to vehicle-treated A53T mice (Fig. 8D, E, F). Overall, these results suggest that oral administration of NaB can increase motor functions as well as improve cognitive performance in A53T mice.

Figure 8. Oral feeding of NaB improves locomotor and cognitive functions in aged A53T mice.

Figure 8.

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A53T mice were treated with NaB (50 mg/Kg/day) for two months followed by monitoring locomotor activities by rotarod (A) and pole test (B, pole climb down; C, pole turn) and spatial learning and memory by Barnes maze (D, heat map; E, latency to goal box; F, error). All data are represented as mean ±SEM of six mice per group. One way ANOVA followed by Dunnett’s multiple comparison test was used for statistical analysis; * p < 0.05; ** p < 0.01; *** p < 0.001.

Cinnamon and NaB treatments upregulate neuroprotective proteins like Parkin and DJ-1 in the nigra of aged A53T mice

Since upregulation of Parkin and DJ-1 is known to reduce α-synucleinopathy (Chung et al., 2003; Ulusoy and Kirik, 2008; Kahle et al., 2009) and we found decrease in α-synucleinopathy by cinnamon and NaB, we investigated status of Parkin and DJ-1. Immunoblot analysis showed a remarkable decrease in the protein levels of Parkin and DJ-1 in the nigra of aged A53T mice as compared to non-Tg mice (Fig. 9A–F). However, oral administration of cinnamon significantly upregulated Parkin and DJ-1 in the nigra of aged A53T mice (Fig. 9A–C). Similar to cinnamon treatment, oral NaB also upregulated the expression of Parkin and DJ-1 in the nigra of aged A53T mice (Fig. 9D–F). These results are consistent to our previous finding in MPTP-mouse model of PD (Khasnavis and Pahan, 2012, 2014).

Figure 9. Oral administration of cinnamon and its metabolite NaB upregulates Parkin and DJ-1 in vivo in the nigra of aged A53T mice.

Figure 9.

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Ten months aged A53T mice were oral gavaged with cinnamon (100 mg/Kg/day), NaB (50 mg/Kg/day) and vehicle (0.5% methylcellulose) for two months followed by immunoblot analysis of nigral extracts for Parkin and DJ-1 (A, cinnamon; D, NaB). Actin was run as loading control. Bands were scanned and presented as relative to actin (B, Parkin-cinnamon; C, DJ-1-cinnamon; E, Parkin-NaB; F, DJ-1-NaB). Data represented as mean ± SEM of 4 mice per group. All statistical analysis was performed by student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Discussion

Although the clinical manifestations of primary α-synucleinopathies (PD, DLB and MSA) are heterogeneous, ranging from movement impairment to dementia, these neurodegenerative disorders share the abnormal accumulation of α-syn in proteinaceous inclusions (Villar-Pique et al., 2016). A direct role for misfolded and undegraded α-syn in the pathogenesis of PD was demonstrated by genetic evidence whereby a mutation (A53T) was identified in the α-syn gene in kindred with autosomal dominant PD (Polymeropoulos et al., 1997). Misfolding and aggregation of α-syn has been associated with impairment of proteasomal degradation, another common trait of PD pathogenesis (Snyder et al., 2003; Lindersson et al., 2004). Mechanisms that might control α-syn aggregation in PD are not clear, but may include transcription factor dysregulation (Scherzer et al., 2008). α-syn in LB aggregates is characteristically hyper phosphorylated at serine 129, (Anderson et al., 2006; Oueslati, 2016) and this accounts for more than 90% of the α-syn found in LB (Hasegawa et al., 2002). Hence any strategy to reduce the accumulation of the phosphorylated forms of α-syn will be helpful in halting in progression of synucleinopathies.

Cinnamon bark from cinnamon tree has a long known history as a medicine since medieval times when physicians used it to treat a variety of disorders including arthritis, coughing, hoarseness, sore throats, etc. One of the main components in cinnamon is cinnamaldehyde, which gets converted to cinnamic acid in the liver through oxidation and the cinnamic acid in turn is β-oxidized to benzoate that exists as sodium benzoate (NaB) or benzoyl-CoA (Pahan and Pahan, 2015). Previously we reported that oral administration of ground cinnamon increased the level of NaB in serum and brain of mice and that NaB is readily able to cross the blood-brain barrier (Jana et al., 2013). Here, we also found increase in NaB in the hippocampus of A53T mice upon oral feeding of cinnamon. Interestingly, we demonstrated that oral administration of ground cinnamon and NaB attenuated α-syn pathology in SNpc, hippocampus and brain stem of aged A53T mice, thus leading to the improvement in locomotor and cognitive functions. Therefore, administration of this natural compound may be beneficial in countering α-syn toxicity in Lewy body diseases.

Microglial activation and associated neuroinflammation is one of the prominent pathological features of different neurodegenerative disorders including α-synucleinopathy. Duffy et al (Duffy et al., 2018) have demonstrated that LB-like α-syn inclusions trigger microglial activation prior to nigral degeneration. According to Sarkar et al (Sarkar et al., 2020), Kv1.3, a voltage-gated potassium channel, is transcriptionally upregulated in microglia in response to aggregated α-syn to stimulate neuroinflammation and neurodegeneration. Accordingly, stimulation of inflammation exacerbates α-syn toxicity and neuropathology in Parkinson’s models (La Vitola et al., 2020) and attenuation of microglial activation by minocycline rescues nigrostriatal dopaminergic neurodegeneration caused by mutant α-syn overexpression (Wang et al., 2020). It has been demonstrated that microglia clear neuron-released α-syn (Choi et al., 2020b) and that the disruption of microglial autophagy causes accumulation of misfolded α-syn and loss of dopaminergic neurons, two hallmarks of PD (Choi et al., 2020a). Neuroinflammation and autophagy dysfunction are closely related and it is known chronic microglial inflammation inhibits microglial autophagy and phagocytosis (Plaza-Zabala et al., 2017; Strohm and Behrends, 2020). Therefore, suppression of microglial inflammation is beneficial for α-synucleinopathy and it is nice to see that oral cinnamon is capable of decreasing both microglial and astroglial activation in the nigra of A53T mice.

Parkin, an E3 ubiquitin-protein ligase with multiple neuroprotective functions, is found in LB (Chung et al., 2003; Ulusoy and Kirik, 2008). While Parkin modulates metabolic turnover, it is also known to reduce the aggregation and toxicity of α-syn. Post-translational modification such as Ser129 phosphorylation, nitration, etc. have been shown to enhance the oligomerization of α-syn. The toxicity of α-syn is increased through phosphorylation at serine residues Ser 87 and Ser 129. Interestingly, Parkin overexpression causes the activation of protein phosphatase 2A (PP2A), resulting in de-phosphorylation of α-syn at Ser 87 and Ser 129 and decrease in α-syn-induced cell death and inflammation (Khandelwal et al., 2010). Oxidation of methionine residues of α-syn is known to promote the formation of α-syn oligomers to increase α-syn toxicity (Leong et al., 2009). According to Yu et al (Yu et al., 2010), nitration of α-syn augments toxicity towards dopaminergic neurons. It has been shown that increased α-syn nitration at Tyr39 results from stress induced by monoamine oxidase B (MAO-B) (Danielson et al., 2009). Interestingly, Parkin can reduce the nitration of α-syn via reducing MAO-B expression (Jiang et al., 2006). Similar to Parkin, DJ-1 is another neuroprotective molecule that is known to function as redox-sensitive chaperone, antioxidant, transcriptional regulator, protease and protein deglycase (Kahle et al., 2009). It has been reported that DJ-1 interactions with α-syn reduce aggregation and cellular toxicity of α-syn (Zondler et al., 2014). Moreover, DJ-1 deficiency leads to impaired autophagy and reduced alpha-syn phagocytosis by microglia (Nash et al., 2017). Therefore, upregulation of Parkin and DJ-1 is beneficial for the reduction of α-syn pathology and it is important to mention that oral administration of cinnamon upregulates Parkin and DJ-1 in vivo in the nigra of A53T mice.

Administration of a dopamine agonist or levodopa/carbidopa has been the standard treatment for PD (Salat and Tolosa, 2018). However, these drugs basically take care of the symptoms by either supplying or imitating dopamine. With time, these drugs cause a number of side effects including dyskinesia, thus creating more problems for PD patients. Furthermore, prolonged usage of these pharmacological and surgical interventions against PD has major issues in terms of side effects, short life span and increase in blood-brain barrier (BBB) permeability (De Deurwaerdere and Di Giovanni, 2017). On the other hand, cinnamon and NaB have numerous benefits over currently available PD therapies. These are easily available and can be taken orally, the least painful route. Cinnamon being a long time traditional medicine and NaB, a food preservative do not have side effects. Besides, it is also relatively economical compared to other available treatment for existing anti-PD.

In conclusion, our study delineated that oral cinnamon and NaB attenuated α-synucleinopathy in various regions of the brain including the SNpc, hippocampus and brain stem, and improved locomotor and cognitive behaviors in aged A53T mouse model of α-synucleinopathy. Therefore, intake of cinnamon and its metabolite NaB may ameliorate or delay neurodegeneration in PD, DLB, MSA, and other α-synucleinopathies.

Supplementary Material

11481_2020_9955_MOESM1_ESM

Acknowledgements

This study was supported by merit awards (1I01BX003033 and 1I01BX005002) from US Department of Veterans Affairs and a grant from NIH (NS108025). Moreover, Dr. Pahan is the recipient of a Research Career Scientist Award (1IK6 BX004982) from the Department of Veterans Affairs.

Footnotes

Conflict of interests: None

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of a an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

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