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Updated: Aug 15, 2026
Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Microglial NADPH oxidase mediates leucine enkephalin dopaminergic neuroprotection
Liya Qin1, Yuxin Liu, Xun Qian
1Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Abstract:
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.
Insights
Leucine enkephalin (LE) and its analogue DTLE show neuroprotection for dopaminergic neurons at extremely low femtomolar concentrations. These peptides reduce inflammation by inhibiting microglial NADPH oxidase and subsequent TNFalpha expression, crucial for neuroprotection.
Area of Science:
- Neuroscience
- Pharmacology
- Immunology
Background:
- Dopaminergic (DA) neuron degeneration is implicated in neurodegenerative diseases.
- Inflammation, particularly microglial activation, contributes to DA neurotoxicity.
- Leucine enkephalin (LE) is a known endogenous opioid peptide with potential modulatory roles.
Purpose of the Study:
- To investigate the neuroprotective effects of leucine enkephalin (LE) on dopaminergic (DA) neurons.
- To elucidate the anti-inflammatory mechanisms underlying LE's neuroprotection at femtomolar concentrations.
- To compare the neuroprotective potential of LE and its analogue des-tyrosine leucine enkephalin (DTLE).
Main Methods:
- Primary mesencephalic neuron-glia cultures were utilized.
- Lipopolysaccharide (LPS) was used to induce DA neurotoxicity and inflammation.
- DA uptake assays and tyrosine hydroxylase (TH) immunocytochemistry assessed DA neuron viability.
- Superoxide production, TNFalpha mRNA, and TNFalpha protein levels were measured in microglia.
Main Results:
- Femtomolar concentrations of LE and DTLE protected DA neurons from LPS-induced neurotoxicity.
- Both peptides significantly reduced LPS-induced superoxide production in microglia.
- LE and DTLE attenuated LPS-induced TNFalpha mRNA and protein expression in a superoxide-dependent manner.
- Neuroprotection and anti-inflammatory effects were dependent on microglial NADPH oxidase activity.
Conclusions:
- Leucine enkephalin (LE) and des-tyrosine leucine enkephalin (DTLE) exert neuroprotective effects on dopaminergic neurons at femtomolar concentrations.
- The neuroprotection is mediated by the inhibition of oxidative stress via microglial NADPH oxidase.
- Attenuation of ROS-mediated TNFalpha amplification by LE and DTLE contributes to their anti-inflammatory and neuroprotective properties.
