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,β-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. 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-1β 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. 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. 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.

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-β (Aβ)-induced microglia activation and neuronal cell death, as well as their effect on the modulation of Aβ aggregation. Primary cortical microglia and neurons were exposed to two different populations of Aβ42 oligomers (Aβ42Os) where the oligomeric state had been assigned by capillary electrophoresis and ultrafiltration. When stimulated with high molecular weight Aβ42Os, microglia released proinflammatory cytokines that led to early neuronal cell death. The studied compounds exerted an anti-inflammatory effect on high molecular weight Aβ42O-stimulated microglia and possibly inhibited microglia-mediated neuronal cell toxicity. Furthermore, the tested compounds demonstrated antioligomeric activity during the process of in vitro Aβ42 aggregation. These findings could be investigated further and used for the optimization of multipotent candidate molecules for AD treatment

Serum amyloid A1 (SAA1) is an inducible acute-phase protein produced in the liver and released in response to environmental and inflammatory stimuli, making it a reliable clinical biomarker of acute inflammation. In addition to its hepatic origin, SAA1 has been detected in axonal myelin sheaths in multiple sclerosis and Alzheimer's disease, suggesting that it may influence cells of the central nervous system (CNS) through several receptors, including Toll like receptors (TLRs). We aimed to clarify the role of TLR4 in mediating SAA1-induced inflammation in CNS cells and to elucidate the molecular mechanisms of SAA1 interaction with the TLR4/MD-2 complex. We used an engineered TLR4 reporter cell system to assess TLR4 activation by SAA1. In primary microglia cultures, we evaluated the effects of SAA1 on pro-inflammatory mediators, with or without pharmacological inhibitors, including the TLR4 inhibitor CLI-095, curcumin, and L48H37. Biochemical and computational approaches were used to investigate direct interactions between SAA1 and the TLR4/MD-2 complex. SAA1 activates the TLR4/MD-2 complex in a concentration-dependent manner. This effect was abolished by the TLR4 inhibitor CLI-095. Curcumin and L48H37, which bind to the hydrophobic pocket of MD-2, reduced SAA1-induced release of interleukin-1β, tumor necrosis factor-α, and nitric oxide, as well as NF-κB activation in microglia. Biochemical and computational studies confirmed a direct interaction between SAA1 and TLR4/MD-2. SAA1 directly engages TLR4/MD-2 to trigger neuroinflammatory responses. Targeting this axis may attenuate SAA1-driven neuroinflammation and offers potential therapeutic strategies for neurodegenerative disorders.

In this study we investigated the performance of two norbormide (NRB)-derived fluorescent probes, NRB ^MC009 (green) and NRB ^ZLW0047 (red), on dissected, living larvae of Drosophila , to verify their potential application in confocal microscopy imaging in vivo . To this end, larval tissues were exposed to NRB probes alone or in combination with other commercial dyes or GFP-tagged protein markers. Both probes were rapidly internalized by most tissues (except the central nervous system) allowing each organ in the microscope field to be readily distinguished at low magnification. At the cellular level, the probes showed a very similar distribution (except for fat bodies), defined by loss of signal in the nucleus and plasma membrane, and a preferential localization to endoplasmic reticulum (ER) and mitochondria. They also recognized ER and mitochondrial phenotypes in the skeletal muscles of fruit fly models that had loss of function mutations in the atlastin and mitofusin genes, suggesting NRB ^MC009 and NRB ^ZLW0047 as potentially useful in vivo screening tools for characterizing ER and mitochondria morphological alterations. Feeding of larvae and adult Drosophilae with the NRB-derived dyes led to staining of the gut and its epithelial cells, revealing a potential role in food intake assays. In addition, when flies were exposed to either dye over their entire life cycle no apparent functional or morphological abnormalities were detected. Rapid internalization, a bright signal, a compatibility with other available fluorescent probes and GFP-tagged protein markers, and a lack of toxicity make NRB ^ZLW0047 and, particularly, NRB ^MC009 one of the most highly performing fluorescent probes available for in vivo microscopy studies and food intake assay in Drosophila .