Microglial NADPH Oxidase Mediates Leucine Enkephalin Dopaminergic Neuroprotection

A bstract : Here, we report that leucine enkephalin (LE) is neuroprotective to dopaminergic (DA) neurons at femtomolar concentrations through anti‐inflammatory properties. Mesencephalic neuron‐glia cultures pretreated with femtomolar concentrations of LE (10 −15 ‐10 −13 M) protected DA neurons from lipopolysaccharide (LPS)‐induced DA neurotoxicity, as determined by DA uptake assay and tyrosine hydroxylase (TH) immunocytochemistry (ICC). However, des‐tyrosine leucine enkephalin (DTLE), an LE analogue that is missing the tyrosine residue required for binding to the kappa opioid receptor, was also neuroprotective (10 −15 ‐10 −13 M), as determined by DA uptake assay and TH ICC. Both LE and DTLE (10 −15 ‐10 −13 M) reduced LPS‐induced superoxide production from microglia‐enriched cultures. Further, both LE and DTLE (10 −14 , 10 −13 M) reduced the LPS‐induced tumor necrosis factor‐alpha (TNFα) mRNA and TNFα protein from PHOX +/+ microglia, as determined by quantitative real‐time RT‐PCR and ELISA analysis in mesencephalic neuron‐glia cultures, respectively. However, both peptides failed to inhibit TNFα expression in PHOX −/− cultures, which are unable to produce extracellular superoxide in response to LPS. Additionally, LE and DTLE (10 −14 , 10 −13 M) failed to show any neuroprotection against LPS in PHOX −/− cultures. Together, these data indicate that LE and DTLE are neuroprotective at femtomolar concentrations through the inhibition of oxidative insult associated with microglial NADPH oxidase and the attenuation of the ROS‐mediated amplification of TNFα gene expression in microglia.