Formyl-methionyl-leucyl-phenylalanine-induced dopaminergic neurotoxicity via microglial activation: a mediator between peripheral infection and neurodegeneration?

Abstract

Background: Parkinson disease (PD), a chronic neurodegenerative disease, has been proposed to be a multifactorial disorder resulting from a combination of environmental mechanisms (chemical, infectious, and traumatic), aging, and genetic deficits. Microglial activation is important in the pathogenesis of PD.

Objectives: We investigated dopaminergic (DA) neurotoxicity and the underlying mechanisms of formyl-methionyl-leucyl-phenylalanine (fMLP), a bacteria-derived peptide, in relation to PD.

Methods: We measured DA neurotoxicity using a DA uptake assay and immunocytochemical staining (ICC) in primary mesencephalic cultures from rodents. Microglial activation was observed via ICC, flow cytometry, and superoxide measurement.

Results: fMLP can cause selective DA neuronal loss at concentrations as low as 10(-13) M. Further, fMLP (10(-13) M) led to a significant reduction in DA uptake capacity in neuron/glia (N/G) cultures, but not in microglia-depleted cultures, indicating an indispensable role of microglia in fMLP-induced neurotoxicity. Using ICC of a specific microglial marker, OX42, we observed morphologic changes in activated microglia after fMLP treatment. Microglial activation after fMLP treatment was confirmed by flow cytometry analysis of major histocompatibility antigen class II expression on a microglia HAPI cell line. Mechanistic studies revealed that fMLP (10(-13) M)-induced increase in the production of extracellular superoxide from microglia is critical in mediating fMLP-elicited neurotoxicity. Pharmacologic inhibition of NADPH oxidase (PHOX) with diphenylene-iodonium or apocynin abolished the DA neurotoxicity of fMLP. N/G cultures from PHOX-deficient (gp91PHOX-/ -) mice were also insensitive to fMLP-induced DA neurotoxicity.

Conclusion: fMLP (10(-13) M) induces DA neurotoxicity through activation of microglial PHOX and subsequent production of superoxide, suggesting a role of fMLP in the central nervous system inflammatory process.

Keywords: NADPH oxidase; fMLP; inflammation; microglia; neurotoxicity.

Figure 1

Pharmacophore analysis between fMLP and substance P. Similar chemical features shared by these two peptides are illustrated by three-dimensional relationships with the highest fit values. fMLP resembles the amino acid sequence (Phe-Phe-Gly-Leu-Met) of the C-terminus of substance P. Hydrogen bond acceptor (HBA), green; negative ionizable, dark blue; hydrophobic, light blue.

Figure 2

Effect of fMLP on rat primary mesencephalic N/G cultures 8 days after treatment with vehicle (control), LPS (5 ng/mL) as a positive control, or different concentrations of fMLP. (A) DA neurotoxicity measured using the [³H]DA uptake assay; values are mean ± SD from four independent experiments in triplicate. (B) GABA neurotoxicity measured using the [³H]GABA uptake assay; values are mean ± SD from three independent experiments in triplicate. (C) Effect of fMLP (10⁻¹³ M) on dopaminergic neurons observed by immunocytochemistry staining with antibody against TH; DA neurotoxicity was measured by counting TH-IR neurons 8 days after treatment. Values are mean ± SD from three independent experiments in triplicate. (D) Representative images shown from three separate experiments for TH-IR neurons in a control culture and in cultures treated with LPS and fMLP. Bar = 50 μm. *p < 0.05 compared with control.

Figure 3

Effects of subpicomolar fMLP on microglial activation in primary rat N/G cultures (LME–) or microglia-depleted cultures (LME+) treated with vehicle, LPS (5 ng/mL; positive control for microglia activation), or fMLP (10⁻¹³ M). (A) DA neurotoxicity measured using the [³H]DA uptake assay 8 days after treatment; values are mean ± SD from three independent experiments in triplicate. (B) Activation of microglia visualized by immunocytochemical staining using OX-42 antibody 24 hr after LPS (5 ng/mL) or fMLP (10⁻¹³ M) treatment in rat mecencephalic N/G cultures. Representative images are shown from three separate experiments; bar = 50 μm. (C, D) Expression of MHCII (OX-6) in the HAPI rat microglial line treated with LPS (10 ng/mL) or fMLP (10⁻¹³ M) measured by flow cytometry (C) and by fluorescence compared with isotype-matched controls (D); values are mean ± SD from three independent experiments. (E) Production of extra-cellular superoxide in N/G cultures from rats treated with vehicle, LPS (10 ng/mL), or fMLP (10⁻¹³ M) and measured by the SOD-inhibitable reduction of WST-1; values are mean ± SD from five independent experiments in triplicate. *p < 0.05 compared with control cultures.

Figure 4

Role of microglial PHOX in subpicomolar fMLP-induced DA neurotoxicity in mesencephalic rat primary N/G cultures. (A) DA neurotoxicity determined by the [³H]DA uptake assay in mesencephalic N/G cultures 8 days after pretreatment with DPI (10⁻⁹ M) or apocynin (0.2 mM) for 30 min and treatment with fMLP (10⁻¹³ M); values are mean ± SD of at least three separate experiments in triplicate. (B) DA neurotoxicity determined by the [³H]DA uptake assay in mesencephalic N/G cultures from gp91^PHOX+/+ and gp91^{PHOX−/ −} mice treated with vehicle or fMLP (10⁻¹³ M) for 8 days; values are mean ± SD from three independent experiments in triplicate. (C) Activation of microglia visualized by immunostaining of Iba-1 antigen in mesencephalic N/G cultures from gp91^PHOX+/+ and gp91^{PHOX−/ −} mice treated with vehicle, LPS (5 ng/mL), or fMLP (10⁻¹³ M) for 24 hr. Images are representative of three independent experiments; bar = 50 μm. *p < 0.05 compared with control cultures. **p < 0.05 compared with fMLP-alone-treated cultures.