Cinnamein Inhibits the Induction of Nitric Oxide and Proinflammatory Cytokines in Macrophages, Microglia and Astrocytes.

Chronic inflammation driven by proinflammatory cytokines (TNFα, IL-1ß, IL-6, etc.), and nitric oxide (NO) plays an important role in the pathogenesis of several autoimmune, inflammatory as well as neurodegenerative disorders like rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, etc. Therefore, identification of nontoxic anti-inflammatory drugs may be beneficial for these autoimmune, inflammatory and neurodegenerative disorders. Cinnamein, an ester derivative of cinnamic acid and benzyl alcohol, is used as a flavoring agent and for its antifungal and antibacterial properties. This study underlines the importance of cinnamein in inhibiting the induction of proinflammatory molecules in RAW 264.7 macrophages and primary mouse microglia and astrocytes. Stimulation of RAW 264.7 macrophages with lipopolysaccharide (LPS) and interferon γ (IFNγ) led to marked production of NO. However, cinnamein pretreatment significantly inhibited LPS- and IFNγ-induced production of NO in RAW 264.7 macrophages. Cinnamein also reduced the mRNA expression of inducible nitric oxide synthase (iNOS) and TNFα in RAW cells. Accordingly, LPS and viral double-stranded RNA mimic polyinosinic: polycytidylic acid (polyIC) stimulated the production of TNFα, IL-1ß and IL-6 in primary mouse microglia, which was inhibited by cinnamein pretreatment. Similarly, cinnamein also inhibited polyIC-induced production of TNFα and IL-6 in primary mouse astrocytes. These results suggest that cinnamein may be used to control inflammation in different autoimmune, inflammatory and neurodegenerative disorders.

Treadmill workout activates PPARα in the hippocampus to upregulate ADAM10, decrease plaques and improve cognitive functions in 5XFAD mouse model of Alzheimer's disease.

Although liver is rich in peroxisome proliferator-activated receptor α (PPARα), recently we have described the presence of PPARα in hippocampus where it is involved in non-amyloidogenic metabolism of amyloid precursor protein (APP) via ADAM10, decreasing amyloid plaques and improving memory and learning. However, mechanisms to upregulate PPARα in vivo in the hippocampus are poorly understood. Regular exercise has multiple beneficial effects on human health and here, we describe the importance of regular mild treadmill exercise in upregulating PPARα in vivo in the hippocampus of 5XFAD mouse model of Alzheimer's disease. We also demonstrate that treadmill exercise remained unable to stimulate ADAM10, reduce plaque pathology and improve cognitive functions in 5XFADΔPPARα mice (5XFAD mice lacking PPARα). On the other hand, treadmill workout increased ADAM10, decreased plaque pathology and protected memory and learning in 5XFADΔPPARß mice (5XFAD mice lacking PPARß). Moreover, the other PPAR (PPARγ) also did not play any role in the transcription of ADAM10 in vivo in the hippocampus of treadmill exercised 5XFAD mice. These results underline an important role of PPARα in which treadmill exercise remains unable to exhibit neuroprotection in the hippocampus in the absence of PPARα.

Glyceryl tribenzoate: A food additive with unique properties to be a substitute for cinnamon.

Cinnamon is a regularly used natural seasoning and flavoring material throughout the world for eras. Recent laboratory studies have demonstrated that oral cinnamon may be beneficial for different neuroinflammatory and neurodegenerative disorders such as multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), and Lewy body diseases (LBD). However, cinnamon's certain limitations (e.g. unavailability of true Ceylon cinnamon throughout the world, impurities in ground cinnamon, etc.) have initiated an interest among researchers to find an alternate of cinnamon that can potentially deliver the same efficacy in the diseases mentioned above. Glyceryl tribenzoate (GTB) is a U.S. Food and Drug Administration (FDA)-approved flavoring ingredient that is used in food and food packaging industries. It has been found that similar to cinnamon, oral GTB is capable of upregulating regulatory T cells and suppressing the autoimmune disease process of experimental autoimmune encephalomyelitis, an animal model of MS. Moreover, both GTB and cinnamon metabolite sodium benzoate (NaB) have the potency to attenuate neurodegenerative pathology in a mouse model of Huntington disease (HD). Here, we have also demonstrated anti-inflammatory property of GTB in astrocytes and macrophages, a property that is also seen with cinnamon and its metabolite sodium benzoate (NaB). Therefore, here, we have made a sincere attempt to discuss the similarities and dissimilarities between cinnamon and GTB with a focus whether GTB has the potential to be considered as a substitute of cinnamon for neuroinflammatory and neurodegenerative disorders.

Upregulation of IL-1 Receptor Antagonist by Aspirin in Glial Cells via Peroxisome Proliferator-Activated Receptor-Alpha.

BACKGROUND: Neuroinflammation is a recognized aspect of Alzheimer's disease (AD) and other neurological illnesses. Interleukin 1 receptor antagonist (IL-1Ra) is an anti-inflammatory molecule, which inhibits inflammatory molecules in different cells including brain cells. However, mechanisms for upregulating IL-1Ra in brain cells are poorly understood. OBJECTIVE: Since aspirin is a widely available pain reliever that shows promise beyond its known pain-relieving capacity, we examined whether aspirin could upregulate the IL-1Ra in the brain. METHODS: We employed PCR, real-time PCR, western blot, immunostaining, chromatin immunoprecipitation (ChIP), and lentiviral transduction in glial cells. 5xFAD mice, an animal model of AD, were treated with aspirin orally via gavage. RESULTS: Aspirin increased the expression of IL-1Ra mRNA and protein in primary mouse astrocytes and mouse BV-2 microglial cells. While investigating the mechanism, we found that the IL-1Ra gene promoter harbors peroxisome proliferator response element (PPRE) and that aspirin upregulated IL-1Ra in astrocytes isolated from peroxisome proliferator-activated receptor-beta knockout (PPARß-/-), but not PPARα-/-, mice. Moreover, we observed that aspirin bound to tyrosine 314 residue of PPARα to stimulate IL-1Ra and that aspirin treatment also increased the recruitment of PPARα to the IL-1Ra promoter. Accordingly, aspirin increased IL-1Ra in vivo in the brain of wild type and PPARß-/-, but not in PPARα-/- mice. Similarly, aspirin treatment also increased astroglial and microglial IL-1Ra in the cortex of 5xFAD, but not 5xFAD/PPARα-/- mice. CONCLUSION: Aspirin may reduce the severity of different neurological conditions by upregulating IL-1Ra and reducing the inflammation.

Stimulation of Dopamine Production by Sodium Benzoate, a Metabolite of Cinnamon and a Food Additive.

BACKGROUND: Parkinson's disease (PD) is one of the most important neurodegenerative disorders in human in which recovery of functions could be achieved by improving the survival and function of residual dopaminergic neurons in the substantia nigra pars compacta. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the dopamine (DA) biosynthesis pathway. OBJECTIVE: Earlier our laboratory has shown that sodium benzoate (NaB), a metabolite of cinnamon and an FDA-approved drug against urea cycle disorders and glycine encephalopathy, increases neuroprotective molecules and protects dopaminergic neurons in a mouse model of PD. Here, we examined whether NaB could stimulate the production of DA in dopaminergic neurons. METHODS: We employed PCR, real-time PCR, western blot, immunostaining, and HPLC to study the signature function of dopaminergic neurons. Locomotor functions were monitored in mice by open-field. RESULTS: NaB increased the mRNA and protein expression of TH to produce DA in mouse MN9D dopaminergic neuronal cells. Accordingly, oral feeding of NaB increased the expression of TH in the nigra, upregulated striatal DA, and improved locomotor activities in striatum of normal C57/BL6 and aged A53T-α-syn transgenic mice. Rapid induction of cAMP response element binding (CREB) activation by NaB in dopaminergic neuronal cells and the abrogation of NaB-induced expression of TH by siRNA knockdown of CREB suggest that NaB stimulates the transcription of TH in dopaminergic neurons via CREB. CONCLUSION: These results indicate a new function of NaB in which it may be beneficial in PD via stimulation of DA production from residual dopaminergic neurons.

Aspirin up-regulates suppressor of cytokine signaling 3 in glial cells via PPARα.

Neuroinflammation is being recognized as a hallmark of different neurodegenerative disorders, including Alzheimer's disease. Suppressor of cytokine signaling 3 (SOCS3) is an anti-inflammatory molecule, which is known to inhibit cytokine signaling and inflammatory gene expression in different cells. However, the pathways by which SOCS3 could be up-regulated in brain cells are poorly understood. Aspirin is a widely available pain reliever that is showing promise beyond its known pain-relieving capacity. This study underlines the importance of aspirin in upregulating SOCS3 in astrocytes and microglia. Aspirin increased the expression of Socs3 mRNA and protein in mouse astrocytes and BV-2 microglial cells in both a time- and dose-dependent manner. While investigating the mechanism, we found that Socs3 gene promoter harbors peroxisome proliferator response element and that aspirin up-regulated SOCS3 in astrocytes isolated from PPARß (-/-), but not PPARα (-/-), mice. Accordingly, aspirin increased SOCS3 in vivo in the cortex of wild type and PPARß (-/-), but not PPARα (-/-), mice. Similarly, aspirin treatment increased astroglial and microglial SOCS3 in the cortex of FAD5X, but not FAD5X/PPARα (-/-), mice. Finally, recruitment of PPARα by aspirin to the proximal, but not distal, peroxisome proliferator response element of the Socs3 promoter suggests that aspirin increases the transcription of Socs3 gene via PPARα. This study describes a novel property of aspirin in elevating SOCS3 in glial cells via PPARα and suggests that aspirin may be further considered for therapeutic application in neuroinflammatory and neurodegenerative disorders.

Upregulation of tripeptidyl-peptidase 1 by 3-hydroxy-(2,2)-dimethyl butyrate, a brain endogenous ligand of PPARα: Implications for late-infantile Batten disease therapy.

The late-infantile Batten disease or late-infantile neuronal ceroid lipofuscinosis (LINCL) is an autosomal recessive lysosomal storage disorder caused by mutations in the Cln2 gene leading to deficiency of lysosomal enzyme tripeptidyl peptidase 1 (TPP1). At present, available options for this fatal disorder are enzyme replacement therapy and gene therapy, which are extensively invasive and expensive. Our study demonstrates that 3-hydroxy-(2,2)-dimethyl butyrate (HDMB), a brain endogenous molecule, is capable of stimulating TPP1 expression and activity in mouse primary astrocytes and a neuronal cell line. HDMB activated peroxisome proliferator-activated receptor-α (PPARα), which, by forming heterodimer with Retinoid X receptor-α (RXRα), transcriptionally upregulated the Cln2 gene. Moreover, by using primary astrocytes from wild type, PPARα-/- and PPARß-/- mice, we demonstrated that HDMB specifically required PPARα for inducing TPP1 expression. Finally, oral administration of HDMB to Cln2 heterozygous (Cln2+/-) mice led to a marked upregulation of TPP1 expression in the motor cortex and striatum in a PPARα-dependent fashion. Our study suggests that HDMB, a brain endogenous ligand of PPARα, might have therapeutic importance for LINCL treatment.

Low-Dose Aspirin Upregulates Tyrosine Hydroxylase and Increases Dopamine Production in Dopaminergic Neurons: Implications for Parkinson's Disease.

Increasing the function of residual dopaminergic neurons in the nigra of PD patients is an important area of research as it may eventually compensate the loss. Although tyrosine hydroxylase (TH) is the rate-limiting enzyme in the dopamine (DA) biosynthesis pathway, there are no effective drugs/molecules to upregulate TH and increase the production of DA in nigral dopaminergic neurons. This study underlines the importance of aspirin in stimulating the expression of TH and increasing the level of DA in dopaminergic neurons. At low doses, aspirin increased the expression of TH and the production of DA in mouse MN9D dopaminergic neuronal cells. Accordingly, oral administration of aspirin increased the expression of TH in the nigra and upregulated the level of DA in striatum of normal C57/BL6 mice and aged A53T α-syn transgenic mice. Oral aspirin also improved locomotor activities of normal mice and A53T transgenic mice. While investigating mechanisms, we found the presence of cAMP response element (CRE) in the promoter of TH gene and the rapid induction of cAMP response element binding (CREB) activation by aspirin in dopaminergic neuronal cells. Aspirin treatment also increased the level of phospho-CREB in the nigra of C57/BL6 mice. The abrogation of aspirin-induced expression of TH by siRNA knockdown of CREB and the recruitment of CREB to the TH gene promoter by aspirin suggest that aspirin stimulates the transcription of TH in dopaminergic neurons via CREB. These results highlight a new property of aspirin in stimulating the TH-DA pathway, which may be beneficial in PD patients. Graphical Abstract ᅟ.

Dietary lysophosphatidylcholine-EPA enriches both EPA and DHA in the brain: potential treatment for depression.

EPA and DHA protect against multiple metabolic and neurologic disorders. Although DHA appears more effective for neuroinflammatory conditions, EPA is more beneficial for depression. However, the brain contains negligible amounts of EPA, and dietary supplements fail to increase it appreciably. We tested the hypothesis that this failure is due to absorption of EPA as triacylglycerol, whereas the transporter at the blood-brain barrier requires EPA as lysophosphatidylcholine (LPC). We compared tissue uptake in normal mice gavaged with equal amounts (3.3 µmol/day) of either LPC-EPA or free EPA (surrogate for current supplements) for 15 days and also measured target gene expression. Compared with the no-EPA control, LPC-EPA increased brain EPA >100-fold (from 0.03 to 4 µmol/g); free EPA had little effect. Furthermore, LPC-EPA, but not free EPA, increased brain DHA 2-fold. Free EPA increased EPA in adipose tissue, and both supplements increased EPA and DHA in the liver and heart. Only LPC-EPA increased EPA and DHA in the retina, and expression of brain-derived neurotrophic factor, cyclic AMP response element binding protein, and 5-hydroxy tryptamine (serotonin) receptor 1A in the brain. These novel results show that brain EPA can be increased through diet. Because LPC-EPA increased both EPA and DHA in the brain, it may help in the treatment of depression as well as neuroinflammatory diseases, such as Alzheimer's disease.

Aspirin ameliorates experimental autoimmune encephalomyelitis through interleukin-11-mediated protection of regulatory T cells.

Multiple sclerosis (MS) is a human disease that results from autoimmune T cells targeting myelin protein that is expressed within the central nervous system. In MS, the number of FoxP3-expressing regulatory T cells (Tregs) is reduced, which facilitates the activation of autoreactive T cells. Because aspirin (acetylsalicylic acid) is the most widely used nonsteroidal anti-inflammatory drug, we examined its immunomodulatory effect in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We found that low-dose aspirin suppressed the clinical symptoms of EAE in mouse models of both relapsing-remitting and chronic disease. Aspirin reduced the development of EAE driven by myelin basic protein (MBP)-specific T cells and the associated perivascular cuffing, inflammation, and demyelination. The effects of aspirin required the presence of CD25+FoxP3+ Tregs Aspirin increased the amounts of Foxp3 and interleukin-4 (IL-4) in T cells and suppressed the differentiation of naïve T cells into T helper 17 (TH17) and TH1 cells. Aspirin also increased the transcription of Il11 mediated by the transcription factor CREB, which was necessary for the generation of Tregs Neutralization of IL-11 negated the effects of aspirin on Treg development and exacerbated EAE. Furthermore, we found that IL-11 alone was sufficient to maintain the percentage of FoxP3+ Tregs and protect mice from EAE. These results identify a previously uncharacterized mode of action of aspirin.

Selective disruption of TLR2-MyD88 interaction inhibits inflammation and attenuates Alzheimer's pathology.

Induction of TLR2 activation depends on its association with the adapter protein MyD88. We have found that TLR2 and MyD88 levels are elevated in the hippocampus and cortex of patients with Alzheimer's disease (AD) and in a 5XFAD mouse model of AD. Since there is no specific inhibitor of TLR2, to target induced TLR2 from a therapeutic angle, we engineered a peptide corresponding to the TLR2-interacting domain of MyD88 (TIDM) that binds to the BB loop of only TLR2, and not other TLRs. Interestingly, WT TIDM peptide inhibited microglial activation induced by fibrillar Aß1-42 and lipoteichoic acid, but not 1-methyl-4-phenylpyridinium, dsRNA, bacterial lipopolysaccharide, flagellin, or CpG DNA. After intranasal administration, WT TIDM peptide reached the hippocampus, reduced hippocampal glial activation, lowered Aß burden, attenuated neuronal apoptosis, and improved memory and learning in 5XFAD mice. However, WT TIDM peptide was not effective in 5XFAD mice lacking TLR2. In addition to its effects in 5XFAD mice, WT TIDM peptide also suppressed the disease process in mice with experimental allergic encephalomyelitis and collagen-induced arthritis. Therefore, selective targeting of the activated status of 1 component of the innate immune system by WT TIDM peptide may be beneficial in AD as well as other disorders in which TLR2/MyD88 signaling plays a role in disease pathogenesis.

Glyceryl Tribenzoate: A Flavoring Ingredient, Inhibits the Adoptive Transfer of Experimental Allergic Encephalomyelitis via TGF-ß: Implications for Multiple Sclerosis Therapy.

Multiple sclerosis (MS) is the most common autoimmune demyelinating disease of the central nervous system (CNS). Here, we have explored a novel use of glyceryl tribenzoate (GTB), a flavoring ingredient, in ameliorating the disease process of experimental allergic encephalomyelitis (EAE), an animal model of MS, via TGF-ß. Oral feeding of GTB suppressed clinical symptoms of adoptively-transferred relapsing-remitting (RR) EAE in recipient mice and suppressed the generation of encephalitogenic T cells in donor mice. GTB also attenuated clinical symptoms of RR-EAE in PLP-TCR transgenic mice and chronic EAE in male C57/BL6 mice. Accordingly, GTB also suppressed perivascular cuffing, preserved the integrity of blood-brain barrier and blood-spinal cord barrier, inhibited inflammation, and stopped demyelination in the CNS of EAE mice. Interestingly, GTB treatment upregulated TGF-ß and enriched regulatory T cells (Tregs) in splenocytes as well as in vivo in EAE mice. Blocking TGF-ß by neutralizing antibodies abrogated GTB-mediated enrichment of Tregs and protection of EAE. These results suggest that oral GTB may be considered as a possible therapy for MS patients.

Cinnamon Converts Poor Learning Mice to Good Learners: Implications for Memory Improvement.

This study underlines the importance of cinnamon, a commonly used natural spice and flavoring material, and its metabolite sodium benzoate (NaB) in converting poor learning mice to good learning ones. NaB, but not sodium formate, was found to upregulate plasticity-related molecules, stimulate NMDA- and AMPA-sensitive calcium influx and increase of spine density in cultured hippocampal neurons. NaB induced the activation of CREB in hippocampal neurons via protein kinase A (PKA), which was responsible for the upregulation of plasticity-related molecules. Finally, spatial memory consolidation-induced activation of CREB and expression of different plasticity-related molecules were less in the hippocampus of poor learning mice as compared to good learning ones. However, oral treatment of cinnamon and NaB increased spatial memory consolidation-induced activation of CREB and expression of plasticity-related molecules in the hippocampus of poor-learning mice and converted poor learners into good learners. These results describe a novel property of cinnamon in switching poor learners to good learners via stimulating hippocampal plasticity.