Microglial NADPH oxidase mediates leucine enkephalin dopaminergic neuroprotection.
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 (TNFalpha) mRNA and TNFalpha 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 TNFalpha 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 TNFalpha gene expression in microglia.
Qin
Liya
L
Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Liu
Yuxin
Y
Qian
Xun
X
Hong
Jau-Shyong
JS
Block
Michelle L
ML
D000818:Animals; D000893:Anti-Inflammatory Agents; D002478:Cells, Cultured; D004298:Dopamine / Q000502:physiology*; D004743:Enkephalin, Leucine / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D008070:Lipopolysaccharides / Q000633:toxicity; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000201:enzymology*; D019255:NADPH Oxidases / Q000235:genetics / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000201:enzymology; D018696:Neuroprotective Agents; D009842:Oligopeptides / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D007202:Indicators and Reagents; D008070:Lipopolysaccharides; D018696:Neuroprotective Agents; D009842:Oligopeptides; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D004743:Enkephalin, Leucine; C037653:deltakephalin; D019255:NADPH Oxidases; D004298:Dopamine