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.

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. 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. 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. 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).

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.

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. 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. 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. 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.

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.

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. 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. 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)-1β, 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. 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.