Functional Selectivity of Coumarin Derivates Acting via GPR55 in Neuroinflammation.

Anti-neuroinflammatory treatment has gained importance in the search for pharmacological treatments of different neurological and psychiatric diseases, such as depression, schizophrenia, Parkinson's disease, and Alzheimer's disease. Clinical studies demonstrate a reduction of the mentioned diseases' symptoms after the administration of anti-inflammatory drugs. Novel coumarin derivates have been shown to elicit anti-neuroinflammatory effects via G-protein coupled receptor GPR55, with possibly reduced side-effects compared to the known anti-inflammatory drugs. In this study, we, therefore, evaluated the anti-inflammatory capacities of the two novel coumarin-based compounds, KIT C and KIT H, in human neuroblastoma cells and primary murine microglia. Both compounds reduced PGE2-concentrations likely via the inhibition of COX-2 synthesis in SK-N-SH cells but only KIT C decreased PGE2-levels in primary microglia. The examination of other pro- and anti-inflammatory parameters showed varying effects of both compounds. Therefore, the differences in the effects of KIT C and KIT H might be explained by functional selectivity as well as tissue- or cell-dependent expression and signal pathways coupled to GPR55. Understanding the role of chemical residues in functional selectivity and specific cell- and tissue-targeting might open new therapeutic options in pharmacological drug development and might improve the treatment of the mentioned diseases by intervening in an early step of their pathogenesis.

Anti-Inflammatory and Anti-Oxidative Effects of AM404 in IL-1ß-Stimulated SK-N-SH Neuroblastoma Cells.

An emerging number of studies address the involvement of neuroinflammation and oxidative stress in the pathophysiology of central nervous system (CNS) disorders such as depression, schizophrenia, anxiety, and neurodegenerative diseases. Different cytokines and molecules, such as prostaglandin (PG) E2, are associated with neuroinflammatory processes. The active acetaminophen metabolite AM404 has been shown to prevent inflammation and neuroinflammation in primary microglia and organotypic hippocampal slice cultures. However, its effects on pathophysiological conditions in the CNS and especially on neurons are still poorly understood. In this study, we therefore evaluated the effects of AM404 and acetaminophen on the arachidonic acid cascade and oxidative stress induced by interleukin (IL)-1ß in human SK-N-SH neuronal cells. We observed that AM404 and acetaminophen significantly and concentration-dependent inhibited IL-1ß-induced release of PGE2, independent of cyclooxygenases (COX)-1 and COX-2 enzymatic activity as well as COX-2 mRNA and protein levels in SK-N-SH-cells. The reduction of IL-1ß-induced PGE2-release by AM404 and acetaminophen treatment might be mediated by the 8-iso-PGF2α pathway since IL-1ß-induced synthesis of this free radical marker is dose-dependently reduced by both compounds, respectively. Therefore, understanding of the potential therapeutic properties of AM404 in neuroinflammation and oxidative stress might lead to future treatment options of different neurological disorders.

Targeting Oxidative Stress: Novel Coumarin-Based Inverse Agonists of GPR55.

Oxidative stress is associated with different neurological and psychiatric diseases. Therefore, development of new pharmaceuticals targeting oxidative dysregulation might be a promising approach to treat these diseases. The G-protein coupled receptor 55 (GPR55) is broadly expressed in central nervous tissues and cells and is involved in the regulation of inflammatory and oxidative cell homeostasis. We have recently shown that coumarin-based compounds enfold inverse agonistic activities at GPR55 resulting in the inhibition of prostaglandin E2. However, the antioxidative effects mediated by GPR55 were not evaluated yet. Therefore, we investigated the antioxidative effects of two novel synthesized coumarin-based compounds, KIT C and KIT H, in primary mouse microglial and human neuronal SK-N-SK cells. KIT C and KIT H show antioxidative properties in SK-N-SH cells as well as in primary microglia. In GPR55-knockout SK-N-SH cells, the antioxidative effects are abolished, suggesting a GPR55-dependent antioxidative mechanism. Since inverse agonistic GPR55 activation in the brain seems to be associated with decreased oxidative stress, KIT C and KIT H possibly act as inverse agonists of GPR55 eliciting promising therapeutic options for oxidative stress related diseases.

Effects of a Novel GPR55 Antagonist on the Arachidonic Acid Cascade in LPS-Activated Primary Microglial Cells.

Neuroinflammation is a crucial process to maintain homeostasis in the central nervous system (CNS). However, chronic neuroinflammation is detrimental, and it is described in the pathogenesis of CNS disorders, including Alzheimer's disease (AD) and depression. This process is characterized by the activation of immune cells, mainly microglia. The role of the orphan G-protein-coupled receptor 55 (GPR55) in inflammation has been reported in different models. However, its role in neuroinflammation in respect to the arachidonic acid (AA) cascade in activated microglia is still lacking of comprehension. Therefore, we synthesized a novel GPR55 antagonist (KIT 10, 0.1-25 µM) and tested its effects on the AA cascade in lipopolysaccharide (LPS, 10 ng / mL)-treated primary rat microglia using Western blot and EIAs. We show here that KIT 10 potently prevented the release of prostaglandin E2 (PGE2), reduced microsomal PGE2 synthase (mPGES-1) and cyclooxygenase-2 (COX-2) synthesis, and inhibited the phosphorylation of Ikappa B-alpha (IκB-α), a crucial upstream step of the inflammation-related nuclear factor-kappaB (NF-κB) signaling pathway. However, no effects were observed on COX-1 and -2 activities and mitogen-activated kinases (MAPK). In summary, the novel GPR55 receptor antagonist KIT 10 reduces neuroinflammatory parameters in microglia by inhibiting the COX-2/PGE2 pathway. Further experiments are necessary to better elucidate its effects and mechanisms. Nevertheless, the modulation of inflammation by GPR55 might be a new therapeutic option to treat CNS disorders with a neuroinflammatory background such as AD or depression.

Repeated bifenthrin exposure alters hippocampal Nurr-1/AChE and induces depression-like behavior in adult rats.

Exposure to insecticides has been associated with depression-like symptoms, especially among occupationally exposed populations, such as farmers. Although the neurotoxicity of pyrethroids such as bifenthrin (BF) is well established, it is still unclear whether exposure to BF may have deleterious effects on the hippocampus and thus behavior. We verified the hypothesis that repeated exposure to BF in a rat model elicits neurochemical and behavioral alterations, the latter of which reflects depression-related symptoms. Adult male Wistar rats (n = 12 per group) were orally administered with different doses of BF (0.6 or 2.1 mg/kg body weight (b.w.)) on a daily basis for 60 days; control rats received the vehicle (corn oil). Different biochemical changes were assessed in the hippocampus, a region of the brain regulating spatial memory; behavioral tests were also conducted. Our results revealed depressive-like behaviors that were expressed by increased despair behavior in the Forced-swimming test. Repeated exposure to BF also decreased acetylcholinesterase (AChE) activity in the hippocampus and butyrylcholinesterase (BuChE) activity in plasma. A significant reduction in the activities of hippocampal membrane-bound ATPases (Na+/K+-ATPase and Mg2+-ATPase) was also observed in BF-treated rats compared to controls. Furthermore, a significant decrease in mRNA expression and protein synthesis of both AChE and orphan nuclear receptor (Nurr-1), as well as in the expression of muscarinic-cholinergic receptor (M1 mAchR) and nicotinic-cholinergic receptor (nAchR2α) was observed in the hippocampus of treated rats compared to controls. Also, BF exposure induced apoptosis as assessed by hippocampal Casp-3 protein levels. Our findings suggest that repeated exposure to BF affects hippocampal signaling and Nurr-1/AChE, and this was accompanied by depression-like state in adult rats.

Neuroprotective Effect of AM404 Against NMDA-Induced Hippocampal Excitotoxicity.

Different studies have demonstrated that inflammation and alterations in glutamate neurotransmission are two events contributing to the pathophysiology of neurodegenerative or neurological disorders. There are evidences that N-arachidonoylphenolamine (AM404), a cannabinoid system modulator and paracetamol metabolite, modulates inflammation and exerts neuroprotective effects on Huntington's (HD) and Parkinson's diseases (PD), and ischemia. However, the effects of AM404 on the production of inflammatory mediators and excitotoxicity in brain tissue stimulated with N-methyl-D-aspartic acid (NMDA) are not elucidated. In this present study, we investigated the effects of AM404 on the production of inflammatory mediators and neuronal cell death induced by NMDA in organotypic hippocampal slices cultures (OHSC) using qPCR, western blot (WB), and immunohistochemistry. Moreover, to comprehend the mechanism of excitotoxicity, we evaluated the effects of AM404 on glutamate release in hippocampal synaptosomes and the NMDA-induced calcium responses in acute hippocampal slices. Our results showed that AM404 led to a significant decrease in cell death induced by NMDA, through a mechanism possibly involving the reduction of glutamate release and the calcium ions responses. Furthermore, it decreased the expression of the interleukin (IL)-1ß. This study provides new significant insights about the anti-inflammatory and neuroprotection effects of AM404 on NMDA-induced excitotoxicity. To understand the effects of AM404 in these processes might contribute to the therapeutic potential of AM404 in diseases with involvement of neuroinflammation and neurodegeneration and might lead to a possible future treatment of neurodegenerative diseases.

Anti-neuroinflammatory effects of GPR55 antagonists in LPS-activated primary microglial cells.

BACKGROUND: Neuroinflammation plays a vital role in Alzheimer's disease and other neurodegenerative conditions. Microglia are the resident mononuclear immune cells of the central nervous system, and they play essential roles in the maintenance of homeostasis and responses to neuroinflammation. The orphan G-protein-coupled receptor 55 (GPR55) has been reported to modulate inflammation and is expressed in immune cells such as monocytes and microglia. However, its effects on neuroinflammation, mainly on the production of members of the arachidonic acid pathway in activated microglia, have not been elucidated in detail. METHODS: In this present study, a series of coumarin derivatives, that exhibit GPR55 antagonism properties, were designed. The effects of these compounds on members of the arachidonic acid cascade were studied in lipopolysaccharide (LPS)-treated primary rat microglia using Western blot, qPCR, and ELISA. RESULTS: We demonstrate here that the various compounds with GPR55 antagonistic activities significantly inhibited the release of PGE2 in primary microglia. The inhibition of LPS-induced PGE2 release by the most potent candidate KIT 17 was partially dependent on reduced protein synthesis of mPGES-1 and COX-2. KIT 17 did not affect any key enzyme involved on the endocannabinoid system. We furthermore show that microglia expressed GPR55 and that a synthetic antagonist of the GPR receptor (ML193) demonstrated the same effect of the KIT 17 on the inhibition of PGE2. CONCLUSIONS: Our results suggest that KIT 17 is acting as an inverse agonist on GPR55 independent of the endocannabinoid system. Targeting GPR55 might be a new therapeutic option to treat neurodegenerative diseases with a neuroinflammatory background such as Alzheimer's disease, Parkinson, and multiple sclerosis (MS).

Role of Microglia TLRs in Neurodegeneration.

Toll-like receptors (TLRs) are a group of receptors widely distributed in the organism. In the central nervous system, they are expressed in neurons, astrocytes and microglia. Although their involvement in immunity is notorious, different articles have demonstrated their roles in physiological and pathological conditions, including neurodegeneration. There is increasing evidence of an involvement of TLRs, especially TLR2, 4 and 9 in neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). In this sense, their expression in microglia might modulate the activity of these cells, which in turn, lead to protective or deleterious effects over neurons and other cells. Therefore, TLRs might mediate the link between inflammation and neurodegenerative diseases. However, further studies have to be performed to elucidate the role of the other TLRs in these diseases and to further prove and confirm the pathophysiological role of all TLRs in neurodegeneration. In this article, we revise and summarize the current knowledge regarding the role of TLRs in neurodegeneration with the focus on the possible functions of these receptors in microglia.

Correction to: AM404, paracetamol metabolite, prevents prostaglandin synthesis in activated microglia by inhibiting COX activity.

After publication of the article [1], it has been brought to our attention that the caption for Figure 2 has been mistakenly replaced with a reproduction of the Figure 4 caption.

AM404, paracetamol metabolite, prevents prostaglandin synthesis in activated microglia by inhibiting COX activity.

BACKGROUND: N-arachidonoylphenolamine (AM404), a paracetamol metabolite, is a potent agonist of the transient receptor potential vanilloid type 1 (TRPV1) and low-affinity ligand of the cannabinoid receptor type 1 (CB1). There is evidence that AM404 exerts its pharmacological effects in immune cells. However, the effect of AM404 on the production of inflammatory mediators of the arachidonic acid pathway in activated microglia is still not fully elucidated. METHOD: In the present study, we investigated the effects of AM404 on the eicosanoid production induced by lipopolysaccharide (LPS) in organotypic hippocampal slices culture (OHSC) and primary microglia cultures using Western blot, immunohistochemistry, and ELISA. RESULTS: Our results show that AM404 inhibited LPS-mediated prostaglandin E2 (PGE2) production in OHSC, and LPS-stimulated PGE2 release was totally abolished in OHSC if microglial cells were removed. In primary microglia cultures, AM404 led to a significant dose-dependent decrease in the release of PGE2, independent of TRPV1 or CB1 receptors. Moreover, AM404 also inhibited the production of PGD2 and the formation of reactive oxygen species (8-iso-PGF2 alpha) with a reversible reduction of COX-1- and COX-2 activity. Also, it slightly decreased the levels of LPS-induced COX-2 protein, although no effect was observed on LPS-induced mPGES-1 protein synthesis. CONCLUSIONS: This study provides new significant insights about the potential anti-inflammatory role of AM404 and new mechanisms of action of paracetamol on the modulation of prostaglandin production by activated microglia.

Neuroprotective effects of intrastriatal injection of rapamycin in a mouse model of excitotoxicity induced by quinolinic acid.

BACKGROUND: The mammalian target of rapamycin (mTOR) is a kinase involved in a variety of physiological and pathological functions. However, the exact role of mTOR in excitotoxicity is poorly understood. Here, we investigated the effects of mTOR inhibition with rapamycin against neurodegeneration, and motor impairment, as well as inflammatory profile caused by an excitotoxic stimulus. METHODS: A single and unilateral striatal injection of quinolinic acid (QA) was used to induce excitotoxicity in mice. Rapamycin (250 nL of 0.2, 2, or 20 µM; intrastriatal route) was administered 15 min before QA injection. Forty-eight hours after QA administration, rotarod test was performed to evaluate motor coordination and balance. Fluoro-Jade C, Iba-1, and GFAP staining were used to evaluate neuronal cell death, microglia morphology, and astrocytes density, respectively, at this time point. Levels of cytokines and neurotrophic factors were measured by ELISA and Cytometric Bead Array 8 h after QA injection. Striatal synaptosomes were used to evaluate the release of glutamate. RESULTS: We first demonstrated that rapamycin prevented the motor impairment induced by QA. Moreover, mTOR inhibition also reduced the neurodegeneration and the production of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α induced by excitotoxic stimulus. The lowest dose of rapamycin also increased the production of IL-10 and prevented the reduction of astrocyte density induced by QA. By using an in vitro approach, we demonstrated that rapamycin differently alters the release of glutamate from striatal synaptosomes induced by QA, reducing or enhancing the release of this neurotransmitter at low or high concentrations, respectively. CONCLUSION: Taken together, these data demonstrated a protective effect of rapamycin against an excitotoxic stimulus. Therefore, this study provides new evidence of the detrimental role of mTOR in neurodegeneration, which might represent an important target for the treatment of neurodegenerative diseases.

Neurodegeneration Alters Metabolic Profile and Sirt 1 Signaling in High-Fat-Induced Obese Mice.

Different factors may contribute to the development of neurodegenerative diseases. Among them, metabolic syndrome (MS), which has reached epidemic proportions, has emerged as a potential element that may be involved in neurodegeneration. Furthermore, studies have shown the importance of the sirtuin family in neuronal survival and MS, which opens the possibility of new pharmacological targets. This study investigates the influence of sirtuin metabolic pathways by examining the functional capacities of glucose-induced obesity in an excitotoxic state induced by a quinolinic acid (QA) animal model. Mice were divided into two groups that received different diets for 8 weeks: one group received a regular diet, and the other group received a high-fat diet (HF) to induce MS. The animals were submitted to a stereotaxic surgery and subdivided into four groups: Standard (ST), Standard-QA (ST-QA), HF and HF-QA. The QA groups were given a 250 nL quinolinic acid injection in the right striatum and PBS was injected in the other groups. Obese mice presented with a weight gain of 40 % more than the ST group beyond acquiring an insulin resistance. QA induced motor impairment and neurodegeneration in both ST-QA and HF-QA, although no difference was observed between these groups. The HF-QA group showed a reduction in adiposity when compared with the groups that received PBS. Therefore, the HF-QA group demonstrated a commitment-dependent metabolic pathway. The results suggest that an obesogenic diet does not aggravate the neurodegeneration induced by QA. However, the excitotoxicity induced by QA promotes a sirtuin pathway impairment that contributes to metabolic changes.

Studying neurodegenerative diseases in culture models.

Neurodegenerative diseases are pathological conditions that have an insidious onset and chronic progression. Different models have been established to study these diseases in order to understand their underlying mechanisms and to investigate new therapeutic strategies. Although various in vivo models are currently in use, in vitro models might provide important insights about the pathogenesis of these disorders and represent an interesting approach for the screening of potential pharmacological agents. In the present review, we discuss various in vitro and ex vivo models of neurodegenerative disorders in mammalian cells and tissues.

Validation of a direct agglutination test prototype kit for the diagnosis of visceral leishmaniasis.

BACKGROUND: A freeze-dried antigen was developed with Leishmania (L.) infantum and used for the production of a prototype direct agglutination test kit for the laboratory diagnosis of visceral leishmaniasis (VL), called DAT-LPC. On this study the diagnosis validity of this prototype was performed. METHODS: To evaluate the sensitivity and specificity 103 samples from Brazilian patients with VL and 110 samples from patients with other parasitic infections, and healthy subjects were assayed with DAT-LPC and DAT-KIT (Royal Tropical Institute, Amsterdam, NL). Additionally, the results of 103 samples of VL patients based on two agglutination tests were transformed in Log10 and correlated. RESULTS: The DAT-LPC showed a sensitivity of 99.0%, specificity of 98.2% and diagnosis validity of 98.6%, which were similar to those found by the DAT-KIT (p > 0.05). Moreover, there was positive correlation between the positive titers obtained by DAT-LPC and by DAT-KIT (Spearman correlation coefficient of 0.75 p = 0.0001). CONCLUSIONS: DAT-LPC showed thermal stability and diagnosis performance similar to those of the DAT-KIT. Our results suggest that DAT-LPC is a robust, simple, equipment-independent and efficient tool for the diagnosis of VL and should thus replace the IFAT as routine diagnostic test in the Brazilian public health system.

Studying neurodegenerative diseases in culture models

Neurodegenerative diseases are pathological conditions that have an insidious onset and chronic progression. Different models have been established to study these diseases in order to understand their underlying mechanisms and to investigate new therapeutic strategies. Although various in vivo models are currently in use, in vitro models might provide important insights about the pathogenesis of these disorders and represent an interesting approach for the screening of potential pharmacological agents. In the present review, we discuss various in vitro and ex vivo models of neurodegenerative disorders in mammalian cells and tissues.

Direct agglutination test (DAT): improvement of biosafety for laboratory diagnosis of visceral leishmaniasis.

In this study, the direct agglutination test (DAT), using 2-mercaptoethanol (2-ME), kaolin or N-acetylcysteine (NAC) as sample diluents, was used to assay 89 samples from visceral leishmaniasis (VL) patients and 130 samples from patients with other diseases and healthy individuals. Maintaining a cut-off of 1:100, the DAT assays with 2-ME, kaolin or NAC presented sensitivities of 94.4%, 95.5% and 100% (P = 0.09) and specificities of 99.2%, 100% and 97.7% (P = 0.17), respectively. Based on these results, we suggest that NAC can be used as a replacement for 2-ME in the DAT, increasing biosafety in the diagnosis of VL.