BACKGROUND AND PURPOSEToll-like receptor 4 (TLR4) plays a key role in the induction of inflammatory responses both in peripheral organs and the CNS. Curcumin exerts anti-inflammatory functions by interfering with LPS-induced dimerization of TLR4-myeloid differentiation protein-2 (MD-2) complex and suppressing pro-inflammatory mediator release. However, the inhibitory mechanism of curcumin remains to be defined. EXPERIMENTAL APPROACHBinding of bis-demethoxycurcumin (GG6) and its cyclized pyrazole analogue (GG9), lacking the 1,3-dicarbonyl function, to TLR4-MD-2 was determined using molecular docking simulations. The effects of these compounds on cytokine release and NF-κB activation were examined by ELISA and fluorescence staining in LPS-stimulated primary microglia. Interference with TLR4 dimerization was assessed by immunoprecipitation in Ba/F3 cells. KEY RESULTSBoth curcumin analogues bound to the hydrophobic region of the MD-2 pocket. However, only curcumin and GG6, both possessing the 1,3-diketone moiety, inhibited LPS-induced TLR4 dimerization, activation of NF-κB and secretion of proinflammatory cytokines in primary microglia. Consistent with the ability of 1,3-diketones to coordinate divalent metal ions, LPS stimulation in a low magnesium environment decreased pro-inflammatory cytokine release and NF-κB p65 nuclear translocation in microglia and decreased TLR4-MD-2 dimerization in Ba/F3 cells. Curcumin and GG6 also significantly reduced cytokine output in contrast to the pyrazole analogue GG9. CONCLUSIONS AND IMPLICATIONSThese results indicate that phenolic 1,3-diketones, with a structural motif able to coordinate magnesium ions, can modulate LPSmediated TLR4-MD-2 signalling. Taken together, these studies identify a previously uncharacterized mechanism involving magnesium, underlying the inflammatory responses to LPS. Abbreviations

Microglia, the brain's resident macrophages, contribute to immune surveillance and the response to disease and injury. These immune cells play a dual role in the nervous system, having both neurotoxic and neuroprotective effects. Activation of microglia results in the production of inflammatory molecules and neurotoxic factors that often cause or contribute to neurodegenerative diseases. Inhibition of neurotoxic microglia activation and consequent inflammatory processes may represent an important therapeutic target. Phosphatidylserine (PS), an aminophospholipid of plasma membranes, and curcumin, the yellow pigment isolated from the rhizome of the turmeric plant, have both been reported to suppress microglial activation by reducing pro-inflammatory mediator production and release. In this study we analyzed the effects of PS, curcumin, and their association on microglial activation induced by the bacterial toxin lipopolysaccharide. Primary rat cortical microglial cells were treated with increasing concentrations of PS-liposomes and curcumin, alone or in combination, and their effects on pro-inflammatory cytokine release from unstimulated and lipopolysaccharide-stimulated microglia were evaluated by enzyme-linked immunosorbent assay. Isobolographic analysis was performed to investigate the effect of PS-liposomes and curcumin combination. PS and curcumin inhibited the release of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α induced by lipopolysaccharide. Furthermore, PS and curcumin in combination exerted a synergistic effect in down-regulating IL-1β release. These results suggest that the association of PS with curcumin could be of potential therapeutic utility against diseases associated with microglial activation.

Animal models of Parkinson's disease are essential to explore pathophysiological hypotheses and to test new treatment options, including neurotrophic factors. Catecholaminergic neurotoxins used to generate such models are 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. These neurotoxins predominantly kill dopaminergic neurons through oxidative damage and mitochondrial failure, although 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine fails to induce a significant dopaminergic neurodegeneration in rats. The present chapter describes a protocol for the 6-hydroxydopamine rat model based on stereotaxic injection performed only unilaterally, which mimics an early-to-mid stage of the disease.

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Dopaminergic neuronal cell degeneration is the principal characteristic feature of the neuropathology of Parkinson's disease. Cultures of mesencephalic neurons are widely used as a source of dopaminergic neurons for the study of mechanisms implicated in dopaminergic cell death and for the evaluation of potential dopaminergic neuroprotective agents, including neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat mesencephalic cell cultures and their application to evaluating the neuroprotective action of brain-derived neurotrophic factor.