The protective effect of lactoferrin on ventral mesencephalon neurons against MPP + is not connected with its iron binding ability

Abstract

Lactoferrin (Lf) can bind to lactoferrin receptor (LfR), leading to iron transport through the plasma membrane. Besides iron transportation, Lf also has antioxidant and anti-inflammatory properties. In the brain, Lf is only synthesized by activated microglia. LfR is present in blood vessels and nigral dopaminergic neurons. Both nigral iron accumulation and microglia activation is believed to be involved in Parkinson's disease (PD), moreover, increased Lf and LfR in dopaminergic neurons were found in PD cases and MPTP-intoxicated mice. How iron influences microglia to release Lf? Does Lf tend to transport iron to dopaminergic neurons leading to cell death or to protect dopaminergic neuron from neurotoxin? In this study, we observed that iron increased Lf synthesis in activated microglia. In ventral mesencephalon neurons, both iron-free Lf (apo-Lf) and iron-saturated Lf (holo-Lf) exerted neuroprotective effects against MPP(+) by mechanisms, believed to enhance the mitochondrial transmembrane potential, improve Cu/Zn-superoxide dismutase activity, increase Bcl-2 expression. Although apo-Lf but not holo-Lf chelated cellular iron, there was no difference between the two types of Lf in the neuroprotection. Our data indicate that iron overload increases the activated microglia releasing Lf. Lf plays protective role on ventral mesencephalon neurons against MPP(+), which is iron-chelating independent.

Figure 1. FAC enhanced Lf release and mRNA expression in activated microglia triggered by MPP⁺.

(A) Activation of microglia with MPP⁺ increased Lf release, which was further enhanced by initial FAC incubation. (B) Lf mRNA levels in MPP⁺ activated microglia was increased compared with control group. Iron load in microglia elevated the Lf mRNA expression compared with MPP⁺ group. Data are presented as fold changes in Lf mRNA expression with treatment vs control in microglia. *P < 0.05, compared with the control; ^#P < 0.05, compared with MPP⁺ group. n = 6.

Figure 2. ΔΨm assessed by flow cytometry in MPP⁺-treated VM neurons with apo-Lf or holo-Lf pretreatment.

Changes in the mitochondrial membrane potential were measured by rhodamine123 using flow cytometry. (A) Representatives of the fluorometric assay on ΔΨm of different groups. Results are shown as FL1-H (fluorescence 1-histogram), setting the gated region M1 and M2 as a marker to observe the changing levels of fluorescence intensity using CellQuest software. Numbers in the M1 gate of the dot-plots show the percentage of apoptotic cells. The ΔΨm was decreased in MPP⁺-treated apoptotic cells and the ratio of M1 area was increased. 100 ng/ml apo-Lf or holo-Lf pretreatment significantly restored the ΔΨm reduction induced by MPP⁺, and the frequency of apoptotic cells was decreased. X mean = mean channel fluorescence of FL-1; Y mean= the number of living cells. (B) Statistical analysis. Data were presented as mean ± S.E.M. of three independent experiments. Fluorescence values of the control were set to 100%. *P < 0.05, compared with the control; ^#P < 0.05, compared with apo-Lf group; ^{^}P < 0.05, compared with holo-Lf group; n = 6.

Figure 3. LfR protein levels in MPP⁺-treated VM neurons with apo-Lf or holo-Lf pretreatment.

(A) Western blots were applied to detect LfR protein levels. LfR expression was increased in apo-Lf or holo-Lf treated VM neurons compared with control group. Apo-Lf or holo-Lf pretreatment increased the expression of LfR in MPP⁺-treated cells. β-actin was used as a loading control. (B) Statistical analysis. Data were presented as the ratio of LfR to β-actin. Each bar represented the mean ± S.E.M. of 6 independent experiments. *P < 0.05, compared with the control. ^#P < 0.05, compared with apo-Lf group; ^{^}P < 0.05, compared with holo-Lf group.

Figure 4. ΔΨm assessed by flow cytometry in VM neurons with blocking LfR.

To block binding of ligands to LfR, VM neurons were treated with Anti-LfR for 1 h at 37 °C. Then, VM neurons were treated with 100 ng/ml apo-Lf or 100 ng/ml holo-Lf for 4 h followed by 100 μmol/L MPP⁺ for 24 h. (A) Representatives of the fluorometric assay on ΔΨm of different groups. Results are shown as FL1-H (fluorescence 1-histogram), setting the gated region M1 and M2 as a marker to observe the changing levels of fluorescence intensity using CellQuest software. Numbers in the M1 gate of the dot-plots show the percentage of apoptotic cells. The ΔΨm was decreased in MPP⁺-treated apoptotic cells and the ratio of M1 area was increased. 100 ng/ml apo-Lf or holo-Lf pretreatment significantly restored the ΔΨm reduction induced by MPP⁺. Blocking LfR significantly attenuated the protective effect of Lf in VM neurons induced by MPP⁺. X mean = mean channel fluorescence of FL-1; Y mean= the number of living cells. (B) Statistical analysis. Data were presented as mean ± S.E.M. of three independent experiments. Fluorescence values of the control were set to 100%. *P < 0.05, compared with the control; ^#P < 0.05, compared with MPP⁺ group; n = 6.

Figure 5. Cu/Zn–SOD protein levels in MPP⁺-treated VM neurons with apo-Lf or holo-Lf pretreatment.

(A) Western blots were applied to detect Cu/Zn–SOD protein levels. Cu/Zn–SOD protein level was down-regulated in VM neurons by MPP⁺. Apo-Lf or holo-Lf increased the expression of Cu/Zn–SOD in VM neurons compared with the control group. Apo-Lf or holo-Lf pretreatment reversed the decrease of Cu/Zn–SOD expression in MPP⁺-treated cells. β-actin was used as a loading control. (B) Statistical analysis. Data were presented as the ratio of Cu/Zn–SOD to β-actin. Each bar represented the mean ± S.E.M. *P < 0.05, compared with the control; ^#P < 0.05, compared with MPP⁺ group; n = 6.

Figure 6. Bcl-2 and Bax protein levels in MPP⁺-treated VM neurons with apo-Lf or holo-Lf pretreatment.

Western blots were applied to detect Bcl-2 and Bax protein levels. (A) Bcl-2 expression was decreased in MPP⁺-treated cells. Both apo-Lf and holo-Lf could elevate Bcl-2 expressions in VM neurons compared with control group. Apo-Lf or holo-Lf pretreatment increased the expression of Bcl-2 in MPP⁺-treated cells. (B) Bax protein levels had no change in different groups. (C) Effects of Lf on Bcl-2/Bax ratio in different groups. Bcl-2/Bax ratio significantly increased due to apo-Lf or holo-Lf treatment in VM neurons. And apo-Lf or holo-Lf pretreatment reversed the decrease of Bcl-2/Bax ratio in MPP⁺ treated group. β-actin was used as a loading control. Data were presented as the ratio of Bcl-2 or Bax to β-actin. Each bar represented the mean ± S.E.M. *P < 0.05, compared with the control; ^#P < 0.05, compared with MPP⁺ group; n = 4.

Figure 7. Apo-Lf but not holo-Lf reduced the cellular LIP.

LIP in VM neurons was determined by the fluorescence intensity of calcein, an indicator of intracellular pool of free iron. The fluorescence intensity represented the mean value of VM neurons at each time point and was presented as the mean ± S.E.M. of six independent experiments. Results were carried out by two-way ANOVA followed by Student-Newman-Keuls test. There was a significant decrease in the fluorescence intensity in neurons treated with MPP⁺+ FAC compared with the control, indicating increased free iron level. Fluorescence intensities increased in apo-Lf+ MPP⁺+FAC or DFO+MPP⁺+FAC groups, indicating decreased free iron levels in these cells. There was no obvious reverse in fluorescent intensity in holo-Lf+ MPP⁺+FAC group, indicating holo-Lf did not change the cellular iron levels (two-way ANOVA, F = 39.835, P < 0.05, apo-Lf+MPP⁺+FAC vs MPP⁺+FAC; P < 0.05, DFO+MPP⁺+FAC vs MPP⁺+FAC; P < 0.05, holo-Lf+MPP⁺+FAC vs apo-Lf+MPP⁺+FAC and DFO+MPP⁺+FAC;P > 0.05, holo-Lf+MPP⁺+FAC vs MPP⁺+FAC).