The Effect of C-Phycocyanin on Microglia Activation Is Mediated by Toll-like Receptor 4.

The blue-green alga Spirulina platensis is rich in phycocyanins, that exhibit a wide range of pharmacological actions. C-phycocyanin (C-PC), in particular, possesses hepatoprotective, nephroprotective, antioxidant, and anticancer effects. Furthermore, several studies have reported both anti- and proinflammatory properties of this pigment. However, the precise mechanism(s) of action of C-PC in these processes remain largely unknown. Therefore, here we explored the C-PC effect in in vitro microglia activation. The effect of C-PC on the expression and release of IL-1ß and TNF-α and the activation of NF-κB was examined in primary microglia by real-time PCR, ELISA, and immunofluorescence. Treatment with C-PC up-regulated the expression and release of IL-1ß and TNF-α. C-PC also promoted the nuclear translocation of the NF-κB transcription factor. Then, to elucidate the molecular mechanisms for the immunoregulatory function of C-PC, we focused on investigating the role of Toll-like receptor 4 (TLR4). Accordingly, several TLR4 inhibitors have been used. Curcumin, ciprofloxacin, L48H37, and CLI-095 that suppresses specifically TLR4 signaling, blocked IL-1ß and TNF-α. Overall, these results indicate the immunomodulatory effect of C-PC in microglia cultures and show for the first time that the molecular mechanism implicated in this effect may involve TLR4 activation.

Modulation of Amyloid ß-Induced Microglia Activation and Neuronal Cell Death by Curcumin and Analogues.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is not restricted to the neuronal compartment but includes important interactions with immune cells, including microglia. Protein aggregates, common pathological hallmarks of AD, bind to pattern recognition receptors on microglia and trigger an inflammatory response, which contributes to disease progression and severity. In this context, curcumin is emerging as a potential drug candidate able to affect multiple key pathways implicated in AD, including neuroinflammation. Therefore, we studied the effect of curcumin and its structurally related analogues cur6 and cur16 on amyloid-ß (Aß)-induced microglia activation and neuronal cell death, as well as their effect on the modulation of Aß aggregation. Primary cortical microglia and neurons were exposed to two different populations of Aß42 oligomers (Aß42Os) where the oligomeric state had been assigned by capillary electrophoresis and ultrafiltration. When stimulated with high molecular weight Aß42Os, microglia released proinflammatory cytokines that led to early neuronal cell death. The studied compounds exerted an anti-inflammatory effect on high molecular weight Aß42O-stimulated microglia and possibly inhibited microglia-mediated neuronal cell toxicity. Furthermore, the tested compounds demonstrated antioligomeric activity during the process of in vitro Aß42 aggregation. These findings could be investigated further and used for the optimization of multipotent candidate molecules for AD treatment.

Monitoring of Spirulina Flakes and Powders from Italian Companies.

Microalgae and microalgae-derived compounds have great potential as supplements in the human diet and as a source of bioactive products with health benefits. Spirulina (Arthrospira platensis (Nordstedt) Gomont, or Spirulina platensis) belongs to the class of cyanobacteria and has been studied for its numerous health benefits, which include anti-inflammatory properties, among others. This work was aimed at comparing some spirulina products available on the Italian market. The commercial products here analyzed consisted of spirulina cultivated and processed with different approaches. Single-component spirulina products in powder and flake form, free of any type of excipient produced from four different companies operating in the sector, have been analyzed. The macro- and micromorphological examination, and the content of pigments, phycobiliproteins, phenols, and proteins have shown differences regarding the morphology and chemical composition, especially for those classes of particularly unstable compounds such as chlorophylls and carotenoids, suggesting a great influence of both culture conditions and processing methods.

A co-ultramicronized palmitoylethanolamide/luteolin composite mitigates clinical score and disease-relevant molecular markers in a mouse model of experimental autoimmune encephalomyelitis.

BACKGROUND: Persistent and/or recurrent inflammatory processes are the main factor leading to multiple sclerosis (MS) lesions. The composite ultramicronized palmitoylethanolamide, an endogenous N-acylethanolamine, combined with the flavonoid luteolin, PEALut, have been found to exert neuroprotective activities in experimental models of spinal and brain injury and Alzheimer disease, as well as a clinical improvement in human stroke patients. Furthermore, PEALut enhances the expression of different myelin proteins in oligodendrocyte progenitor cells suggesting that this composite might have protective effects in MS experimental models. METHODS: The mouse model of experimental autoimmune encephalomyelitis (EAE) based on active immunization with a fragment of myelin oligodendrocyte glycoprotein (MOG35-55) was used. The daily assessment of clinical score and the expression of serum amyloid A (SAA1), proinflammatory cytokines TNF-α, IL-1ß, IFN-γ, and NLRP3 inflammasome, as well as TLR2, Fpr2, CD137, CD3-γ, and TCR-ζ chain, heterodimers that form T cell surface glycoprotein (TCR), and cannabinoid receptors CB1, CB2, and MBP, were evaluated in the brainstem and cerebellum at different postimmunization days (PIDs). RESULTS: Vehicle-MOG35-55-immunized (MOG35-55) mice developed ascending paralysis which peaked several days later and persisted until the end of the experiment. PEALut, given intraperitoneally daily starting on day 11 post-immunization, dose-dependently improved clinical score over the range 0.1-5 mg/kg. The mRNA expression of SAA1, TNF-α, IL-1ß, IFN-γ, and NLRP3 were significantly increased in MOG35-55 mice at 14 PID. In MOG35-55 mice treated with 5 mg /kg PEALut, the increase of SAA1, TNF- α, IL-1ß, and IFN-γ transcripts at 14 PID was statistically downregulated as compared to vehicle-MOG35-55 mice (p < 0.05). The expression of TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 receptors showed a significant upregulation in vehicle-MOG35-55 mice at 14 PID. Instead, CB1 and MBP transcripts have not changed in expression at any time. In MOG/PEALut-treated mice, TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 mRNAs were significantly downregulated as compared to vehicle MOG35-55 mice. CONCLUSIONS: The present results demonstrate that the intraperitoneal administration of the composite PEALut significantly reduces the development of clinical signs in the MOG35-55 model of EAE. The dose-dependent improvement of clinical score induced by PEALut was associated with a reduction in transcript expression of the acute-phase protein SAA1, TNF-α, IL-1ß, IFN-γ, and NLRP3 proinflammatory proteins and TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 receptors.

Ciprofloxacin and levofloxacin attenuate microglia inflammatory response via TLR4/NF-kB pathway.

BACKGROUND: Neuroinflammation is the response of the central nervous system to events that interfere with tissue homeostasis and represents a common denominator in virtually all neurological diseases. Activation of microglia, the principal immune effector cells of the brain, contributes to neuronal injury by release of neurotoxic products. Toll-like receptor 4 (TLR4), expressed on the surface of microglia, plays an important role in mediating lipopolysaccharide (LPS)-induced microglia activation and inflammatory responses. We have previously shown that curcumin and some of its analogues harboring an α,ß-unsaturated 1,3-diketone moiety, able to coordinate the magnesium ion, can interfere with LPS-mediated TLR4-myeloid differentiation protein-2 (MD-2) signaling. Fluoroquinolone (FQ) antibiotics are compounds that contain a keto-carbonyl group that binds divalent ions, including magnesium. In addition to their antimicrobial activity, FQs are endowed with immunomodulatory properties, but the mechanism underlying their anti-inflammatory activity remains to be defined. The aim of the current study was to elucidate the molecular mechanism of these compounds in the TLR4/NF-κB inflammatory signaling pathway. METHODS: The putative binding mode of five FQs [ciprofloxacin (CPFX), levofloxacin (LVFX), moxifloxacin, ofloxacin, and delafloxacin] to TLR4-MD-2 was determined using molecular docking simulations. The effect of CPFX and LVFX on LPS-induced release of IL-1ß and TNF-α and NF-κB activation was investigated in primary microglia by ELISA and fluorescence staining. The interaction of CPFX and LVFX with TLR4-MD-2 complex was assessed by immunoprecipitation followed by Western blotting using Ba/F3 cells. RESULTS: CPFX and LVFX bound to the hydrophobic region of the MD-2 pocket and inhibited LPS-induced secretion of pro-inflammatory cytokines and activation of NF-κB in primary microglia. Furthermore, these FQs diminished the binding of LPS to TLR4-MD-2 complex and decreased the resulting TLR4-MD-2 dimerization in Ba/F3 cells. CONCLUSIONS: These results provide new insight into the mechanism of the anti-inflammatory activity of CPFX and LVFX, which involves, at least in part, the activation of TLR4/NF-κB signaling pathway. Our findings might facilitate the development of new molecules directed at the TLR4-MD-2 complex, a potential key target for controlling neuroinflammation.

Serum amyloid A primes microglia for ATP-dependent interleukin-1ß release.

BACKGROUND: Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves production of acute-phase proteins, including serum amyloid A (SAA). Interleukin-1ß (IL-1ß), a master regulator of neuroinflammation produced by activated inflammatory cells of the myeloid lineage, in particular microglia, plays a key role in the pathogenesis of acute and chronic diseases of the peripheral nervous system and CNS. IL-1ß release is promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with toll-like receptor (TLR) ligands. METHODS: Purified (> 99%) microglia cultured from neonatal rat cortex and cerebellum were first primed with the putative TLR4/TLR2 agonist SAA (recombinant human Apo-SAA) or the established TLR4 agonist lipopolysaccharide (LPS) followed by addition of ATP. Expression of genes for the NLRP3 inflammasome, IL-1ß, tumor necrosis factor-α (TNF-α), and SAA1 was measured by quantitative real-time polymerase chain reaction (q-PCR). Intracellular and extracellular amounts of IL-1ß were determined by ELISA. RESULTS: Apo-SAA stimulated, in a time-dependent manner, the expression of NLRP3, IL-1ß, and TNF-α in cortical microglia, and produced a concentration-dependent increase in the intracellular content of IL-1ß in these cells. A 2-h 'priming' of the microglia with Apo-SAA followed by addition of ATP for 1 h, resulting in a robust release of IL-1ß into the culture medium, with a concomitant reduction in its intracellular content. The selective P2X7R antagonist A740003 blocked ATP-dependent release of IL-1ß. Microglia prepared from rat cerebellum displayed similar behaviors. As with LPS, Apo-SAA upregulated SAA1 and TLR2 mRNA, and downregulated that of TLR4. LPS was less efficacious than Apo-SAA, perhaps reflecting an action of the latter at TLR4 and TLR2. The TLR4 antagonist CLI-095 fully blocked the action of LPS, but only partially that of Apo-SAA. Although the TLR2 antagonist CU-CPT22 was inactive against Apo-SAA, it also failed to block the TLR2 agonist Pam3CSK4. CONCLUSIONS: Microglia are central to the inflammatory process and a major source of IL-1ß when activated. P2X7R-triggered IL-1ß maturation and export is thus likely to represent an important contributor to this cytokine pool. Given that SAA is detected in Alzheimer disease and multiple sclerosis brain, together with IL-1ß-immunopositive microglia, these findings propose a link between P2X7R, SAA, and IL-1ß in CNS pathophysiology.

Bisdemethoxycurcumin and Its Cyclized Pyrazole Analogue Differentially Disrupt Lipopolysaccharide Signalling in Human Monocyte-Derived Macrophages.

Several studies suggest that curcumin and related compounds possess antioxidant and anti-inflammatory properties including modulation of lipopolysaccharide- (LPS-) mediated signalling in macrophage cell models. We here investigated the effects of curcumin and the two structurally unrelated analogues GG6 and GG9 in primary human blood-derived macrophages as well as the signalling pathways involved. Macrophages differentiated from peripheral blood monocytes for 7 days were activated with LPS or selective Toll-like receptor agonists for 24 h. The effects of test compounds on cytokine production and immunophenotypes evaluated as CD80+/CCR2+ and CD206+/CD163+ subsets were examined by ELISA and flow cytometry. Signalling pathways were probed by Western blot. Curcumin (2.5-10 µM) failed to suppress LPS-induced inflammatory responses. While GG6 reduced LPS-induced IκB-α degradation and showed a trend towards reduced interleukin-1ß release, GG9 prevented the increase in proinflammatory CD80+ macrophage subset, downregulation of the anti-inflammatory CD206+/CD163+ subset, increase in p38 phosphorylation, and increase in cell-bound and secreted interleukin-1ß stimulated by LPS, at least in part through signalling pathways not involving Toll-like receptor 4 and nuclear factor-κB. Thus, the curcumin analogue GG9 attenuated the LPS-induced inflammatory response in human blood-derived macrophages and may therefore represent an attractive chemical template for macrophage pharmacological targeting.

Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes.

BACKGROUND: Toll-like receptor (TLR) activation on microglia and astrocytes are key elements in neuroinflammation which accompanies a number of neurological disorders. While TLR activation on glia is well-established to up-regulate pro-inflammatory mediator expression, much less is known about how ligand engagement of one TLR may affect expression of other TLRs on microglia and astrocytes. METHODS: In the present study, we evaluated the effects of agonists for TLR2 (zymosan), TLR3 (polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double-stranded RNA) and TLR4 (lipopolysaccaride (LPS)) in influencing expression of their cognate receptor as well as that of the other TLRs in cultures of rat cortical purified microglia (>99.5 %) and nominally microglia-free astrocytes. Elimination of residual microglia (a common contaminant of astrocyte cultures) was achieved by incubation with the lysosomotropic agent L-leucyl-L-leucine methyl ester (L-LME). RESULTS: Flow cytometric analysis confirmed the purity (essentially 100 %) of the obtained microglia, and up to 5 % microglia contamination of astrocytes. L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction). The three TLR ligands robustly up-regulated gene expression for pro-inflammatory markers (interleukin-1 and interleukin-6, tumor necrosis factor) in microglia and enriched, but not purified, astrocytes, confirming cellular functionality. LPS, zymosan and poly(I:C) all down-regulated TLR4 messenger RNA (mRNA) and up-regulated TLR2 mRNA at 6 and 24 h. In spite of their inability to elaborate pro-inflammatory mediator output, the nominally microglia-free astrocytes (>99 % purity) also showed similar behaviours to those of microglia, as well as changes in TLR3 gene expression. LPS interaction with TLR4 activates downstream mitogen-activated protein kinase and nuclear factor-κB signalling pathways and subsequently causes inflammatory mediator production. The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor. CONCLUSIONS: TLR2 and TLR4 activation in particular, in concert with microglia and astrocytes, comprise key elements in the initiation and maintenance of neuropathic pain. The finding that both homologous (zymosan) and heterologous (LPS, poly(I:C)) TLR ligands are capable of regulating TLR2 gene expression, in particular, may have important implications in understanding the relative contributions of different TLRs in neurological disorders associated with neuroinflammation.

Reply to: "Palmitoylethanolamide: problems regarding micronization, ultra-micronization and additives" Inflammopharmacology DOI:10.1007/s10787-014-0202-3.

This is a reply to a recently published Commentary: "Palmitoylethanolamide: problems regarding micronization, ultra-micronization and additives" Inflammopharmacology DOI: 10.1007/s10787-014-0202-3 , written in relation to our review article: Skaper SD, Facci L, Fusco M, della Valle MF, Zusso M, Costa B, Giusti P (2014) "Palmitoylethanolamide, a naturally occurring disease-modifying agent in neuropathic pain" Inflammopharmacology 22:79-94 DOI: 10.1007/s10787-013-0191-7 . We believe that the Commentary by Kriek contains a number of erroneous statements and misinterpretations of the published scientific/medical literature which our reply shall elaborate on. Further, the writer of the Commentary has a direct connection to a company, JP Russell Science Ltd that sells palmitoylethanolamide. The take-home message of our review remains as originally stated: "Collectively, the findings presented here propose that palmitoylethanolamide merits further consideration as a disease-modifying agent for controlling inflammatory responses and related chronic and neuropathic pain".

Palmitoylethanolamide, a naturally occurring disease-modifying agent in neuropathic pain.

Persistent pain affects nearly half of all people seeking medical care in the US alone, and accounts for at least $80 billion worth of lost productivity each year. Among all types of chronic pain, neuropathic pain stands out: this is pain resulting from damage or disease of the somatosensory nervous system, and remains largely untreatable. With few available treatment options, neuropathic pain represents an area of significant and growing unmet medical need. Current treatment of peripheral neuropathic pain involves several drug classes, including opioids, gabapentinoids, antidepressants, antiepileptic drugs, local anesthetics and capsaicin. Even so, less than half of patients achieve partial relief. This review discusses a novel approach to neuropathic pain management, based on knowledge of: the role of glia and mast cells in pain and neuroinflammation; the body's innate mechanisms to maintain cellular homeostasis when faced with external stressors provoking, for example, inflammation. The discovery that palmitoylethanolamide, a member of the N-acylethanolamine family which is produced from the lipid bilayer on-demand, is capable of exerting anti-allodynic and anti-hyperalgesic effects by down-modulating both microglial and mast cell activity has led to the application of this fatty acid amide in several clinical studies of neuropathic pain, with beneficial outcome and no indication of adverse effects at pharmacological doses. Collectively, the findings presented here propose that palmitoylethanolamide merits further consideration as a disease-modifying agent for controlling inflammatory responses and related chronic and neuropathic pain.

Fentanyl enhances immune cell response through TLR4/MD-2 complex.

Introduction: Opioids have been shown to induce neuroinflammation and immune cell activation, that might contribute to some of the opioid side effects, such as opioid-induced tolerance and paradoxical hyperalgesia. In this context, TLR4/MD-2 complex has been proposed as an off-target site for opioid action. This study was aimed at investigating the effect of fentanyl on lipopolysaccharide (LPS)-induced TLR4/MD-2 activation in rat primary microglia and human monocyte-derived macrophages (MDM). Materials and Methods: The effect of fentanyl was first explored by measuring the expression and release of different proinflammatory mediators in primary rat microglia and human MDM by real-time PCR and ELISA. Then, the involvement of TLR4/MD-2 signaling was investigated studying NF-κB activation in HEK293 cells stably transfected with human TLR4, MD-2, and CD14 genes (HEK-Blue hTLR4 cells) and in human MDM. Results: Fentanyl increased mRNA levels, as well as the LPS-induced secretion of proinflammatory mediators in primary microglia and MDM. Two inhibitors of TLR4/MD-2 signaling, namely the oxazoline derivative of N-palmitoylethanolamine (PEA-OXA) and CLI-095, blocked the production and release of proinflammatory cytokines by microglia stimulated with LPS and fentanyl, suggesting that TLR4/MD-2 could be the target of the proinflammatory activity of fentanyl. Finally, we showed that fentanyl in combination with LPS activated NF-κB signaling in human MDM and in HEK-Blue hTLR4 cells and this effect was blocked by inhibitors of TLR4/MD-2 complex. Discussion: These results provide new insight into the mechanism of the proinflammatory activity of fentanyl, which involves the activation of TLR4/MD-2 signaling. Our findings might facilitate the development of novel inhibitors of TLR4/MD-2 signaling to combine with opioid-based analgesics for effective and safe pain management.

Regulation of postnatal forebrain amoeboid microglial cell proliferation and development by the transcription factor Runx1.

Microglia are the immune cells of the nervous system, where they act as resident macrophages during inflammatory events underlying many neuropathological conditions. Microglia derive from primitive myeloid precursors that colonize the nervous system during embryonic development. In the postnatal brain, microglia are initially mitotic, rounded in shape (amoeboid), and phagocytically active. As brain development proceeds, they gradually undergo a transition to a surveillant nonphagocytic state characterized by a highly branched (ramified) morphology. This ramification process is almost recapitulated in reverse during the process of microglia activation in the adult brain, when surveillant microglia undergo a ramified-to-amoeboid morphological transformation and become phagocytic in response to injury or disease. Little is known about the mechanisms controlling amoeboid microglial cell proliferation, activation, and ramification during brain development, despite the critical role of these processes in the establishment of the adult microglia pool and their relevance to microglia activation in the adult brain. Here we show that the mouse transcription factor Runx1, a key regulator of myeloid cell proliferation and differentiation, is expressed in forebrain amoeboid microglia during the first two postnatal weeks. Runx1 expression is then downregulated in ramified microglia. Runx1 inhibits mouse amoeboid microglia proliferation and promotes progression to the ramified state. We show further that Runx1 expression is upregulated in microglia following nerve injury in the adult mouse nervous system. These findings provide insight into the regulation of postnatal microglia activation and maturation to the ramified state and have implications for microglia biology in the developing and injured brain.

2-Pentadecyl-2-oxazoline inhibits lipopolysaccharide-induced microglia activation interfering with TLR4 signaling.

AIM: 2-Pentadecyl-2-oxazoline (PEA-OXA), the oxazoline derivative of N-palmitoylethanolamine, exerts anti-inflammatory activity; however, very little is known about the molecular mechanisms underlying this effect. Here, we tested the anti-neuroinflammatory effect of PEA-OXA in primary microglia and we also investigated the possible interaction of the molecule with the Toll-like receptor 4 (TLR4)-myeloid differentiation protein-2 (MD-2) complex. MAIN METHODS: The anti-inflammatory effect of PEA-OXA was analyzed by measuring the expression and release of pro-inflammatory mediators in primary microglia by real-time PCR and ELISA, respectively. The effect of PEA-OXA on the activation of TLR4 signaling was assessed using two stably TLR4-transfected cell lines (i.e., HEK-293 and Ba/F3 cells). Finally, the putative binding mode of PEA-OXA to TLR4-MD-2 was investigated by molecular docking simulations. KEY FINDINGS: Treatment with PEA-OXA resulted in the following effects: (i) it down-regulated gene expression of several pro-inflammatory molecules and the secretion of pro-inflammatory cytokines in LPS stimulated microglia cells; (ii) it did not prevent microglia activation after stimulation with TLR2 ligands; (iii) it prevented TLR4/NF-κB activation triggered by LPS in HEK-Blue™ hTLR4 cells; and (iv) it interfered with the binding of LPS to TLR4-MD-2 complex. Furthermore, molecular docking studies suggested that PEA-OXA could bind MD-2 with a 1:3 (MD-2/PEA-OXA) stoichiometry. CONCLUSION: We show for the first time that the anti-neuroinflammatory effect of PEA-OXA involves its activity against TLR4 signaling, making this molecule a valuable tool for the development of new compounds directed to control neuroinflammation via inhibiting TLR4 signaling.

Targeting the multifaceted neurotoxicity of Alzheimer's disease by tailored functionalisation of the curcumin scaffold.

Simultaneous modulation of multifaceted toxicity arising from neuroinflammation, oxidative stress, and mitochondrial dysfunction represents a valuable therapeutic strategy to tackle Alzheimer's disease. Among the significant hallmarks of the disorder, Aß protein and its aggregation products are well-recognised triggers of the neurotoxic cascade. In this study, by tailored modification of the curcumin-based lead compound 1, we aimed at developing a small library of hybrid compounds targeting Aß protein oligomerisation and the consequent neurotoxic events. Interestingly, from in vitro studies, analogues 3 and 4, bearing a substituted triazole moiety, emerged as multifunctional agents able to counteract Aß aggregation, neuroinflammation and oxidative stress. In vivo proof-of-concept evaluations, performed in a Drosophila oxidative stress model, allowed us to identify compound 4 as a promising lead candidate.

Co-Ultramicronized Palmitoylethanolamide/Luteolin-Induced Oligodendrocyte Precursor Cell Differentiation is Associated With Tyro3 Receptor Upregulation.

Remyelination in patients with multiple sclerosis frequently fails, especially in the chronic phase of the disease promoting axonal and neuronal degeneration and progressive disease disability. Drug-based therapies able to promote endogenous remyelination capability of oligodendrocytes are thus emerging as primary approaches to multiple sclerosis. We have recently reported that the co-ultramicronized composite of palmitoylethanolamide and the flavonoid luteolin (PEALut) promotes oligodendrocyte precursor cell (OPC) maturation without affecting proliferation. Since TAM receptor signaling has been reported to be important modulator of oligodendrocyte survival, we here evaluated the eventual involvement of TAM receptors in PEALut-induced OPC maturation. The mRNAs related to TAM receptors -Tyro3, Axl, and Mertk- were all present at day 2 in vitro. However, while Tyro3 gene expression significantly increased upon cell differentiation, Axl and Mertk did not change during the first week in vitro. Tyro3 gene expression developmental pattern resembled that of MBP myelin protein. In OPCs treated with PEALut the developmental increase of Tyro3 mRNA was significantly higher as compared to vehicle while was reduced gene expression related to Axl and Mertk. Rapamycin, an inhibitor of mTOR, prevented oligodendrocyte growth differentiation and myelination. PEALut, administered to the cultures 30 min after rapamycin, prevented the alteration of mRNA basal expression of the TAM receptors as well as the expression of myelin proteins MBP and CNPase. Altogether, data obtained confirm that PEALut promotes oligodendrocyte differentiation as shown by the increase of MBP and CNPase and Tyro3 mRNAs as well as CNPase and Tyro3 immunostainings. The finding that these effects are reduced when OPCs are exposed to rapamycin suggests an involvement of mTOR signaling in PEALut effects.

Carotenoid Extract Derived from Euglena gracilis Overcomes Lipopolysaccharide-Induced Neuroinflammation in Microglia: Role of NF-κB and Nrf2 Signaling Pathways.

Activation of microglia results in the increased production and release of a series of inflammatory and neurotoxic mediators, which play essential roles in structural and functional neuronal damage and in the development and progression of a number of neurodegenerative diseases. The microalga Euglena gracilis (Euglena), rich in vitamins, minerals, and other nutrients, has gained increasing attention due to its antimicrobial, anti-viral, antitumor, and anti-inflammatory activities. In particular, anti-inflammatory properties of Euglena could exert neuroprotective functions in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying the anti-inflammatory effect of Euglena are not fully understood. In this study, we investigated whether Euglena could attenuate microglia activation and we also studied the mechanism of its anti-inflammatory activity. Our results showed that non-cytotoxic concentrations of a Euglena acetone extract (EAE) downregulated the mRNA expression levels and release of pro-inflammatory mediators, including NO, IL-1ß, and TNF-α in LPS-stimulated microglia. EAE also significantly blocked the LPS-induced nuclear translocation of NF-κB p65 subunit and increased the mRNA expression of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1). Furthermore, the release of pro-inflammatory mediators and NF-κB activation were also blocked by EAE in the presence of ML385, a specific Nrf2 inhibitor. Together, these results show that EAE overcomes LPS-induced microglia pro-inflammatory responses through downregulation of NF-κB and activation of Nrf2 signaling pathways, although the two pathways seem to get involved in an independent manner.

Pre- and Early Post-treatment With Arthrospira platensis (Spirulina) Extract Impedes Lipopolysaccharide-triggered Neuroinflammation in Microglia.

Background: Uncontrolled neuroinflammation and microglia activation lead to cellular and tissue damage contributing to neurodegenerative and neurological disorders. Spirulina (Arthrospira platensis (Nordstedt) Gomont, or Spirulina platensis), a blue-green microalga, which belongs to the class of cyanobacteria, has been studied for its numerous health benefits, which include anti-inflammatory properties, among others. Furthermore, in vivo studies have highlighted neuroprotective effects of Spirulina from neuroinflammatory insults in different brain areas. However, the mechanisms underlying the anti-inflammatory effect of the microalga are not completely understood. In this study we examined the effect of pre- and post-treatment with an acetone extract of Spirulina (E1) in an in vitro model of LPS-induced microglia activation. Methods: The effect of E1 on the release of IL-1ß and TNF-α, expression of iNOS, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1), and the activation of NF-κB was investigated in primary microglia by ELISA, real-time PCR, and immunofluorescence. Results: Pre- and early post-treatment with non-cytotoxic concentrations of E1 down-regulated the release of IL-1ß and TNF-α, and the over-expression of iNOS induced by LPS. E1 also significantly blocked the LPS-induced nuclear translocation of NF-κB p65 subunit, and upregulated gene and protein levels of Nrf2, as well as gene expression of HO-1. Conclusions: These results indicate that the extract of Spirulina can be useful in the control of microglia activation and neuroinflammatory processes. This evidence can support future in vivo studies to test pre- and post-treatment effects of the acetone extract from Spirulina.

Runx transcription factors: lineage-specific regulators of neuronal precursor cell proliferation and post-mitotic neuron subtype development.

Runt-related (RUNX) genes encode evolutionarily conserved transcription factors that play essential roles during development and adult tissue homeostasis. RUNX proteins regulate the transition from proliferation to differentiation in a variety of cell lineages. Moreover, they control the diversification of distinct cellular phenotypes in numerous tissues. Alterations of RUNX functions are associated with several cancers and other human pathologies, underscoring the vital roles of these transcription factors in adult organs. Insights into the functions and regulations of mammalian RUNX proteins have been provided mostly by studies of RUNX involvement in mechanisms of hematopoietic and skeletal development and disease. A growing number of recent investigations are revealing new functions for RUNX family members during the development of the mammalian nervous system. This review will discuss recent progress in the study of RUNX protein involvement in mammalian neural development, with emphasis on the differentiation of olfactory, sensory, and motor neuron lineages.

Pharmacogenomic Characterization in Bipolar Spectrum Disorders.

The holistic approach of personalized medicine, merging clinical and molecular characteristics to tailor the diagnostic and therapeutic path to each individual, is steadily spreading in clinical practice. Psychiatric disorders represent one of the most difficult diagnostic challenges, given their frequent mixed nature and intrinsic variability, as in bipolar disorders and depression. Patients misdiagnosed as depressed are often initially prescribed serotonergic antidepressants, a treatment that can exacerbate a previously unrecognized bipolar condition. Thanks to the use of the patient's genomic profile, it is possible to recognize such risk and at the same time characterize specific genetic assets specifically associated with bipolar spectrum disorder, as well as with the individual response to the various therapeutic options. This provides the basis for molecular diagnosis and the definition of pharmacogenomic profiles, thus guiding therapeutic choices and allowing a safer and more effective use of psychotropic drugs. Here, we report the pharmacogenomics state of the art in bipolar disorders and suggest an algorithm for therapeutic regimen choice.

Corrigendum: An Inflammation-Centric View of Neurological Disease: Beyond the Neuron.

[This corrects the article DOI: 10.3389/fncel.2018.00072.].