Fatty Acid-Binding Protein 3 is Critical for α-Synuclein Uptake and MPP+-Induced Mitochondrial Dysfunction in Cultured Dopaminergic Neurons

Abstract

α-Synuclein is an abundant neuronal protein that accumulates in insoluble inclusions in Parkinson's disease and other synucleinopathies. Fatty acids partially regulate α-Synuclein accumulation, and mesencephalic dopaminergic neurons highly express fatty acid-binding protein 3 (FABP3). We previously demonstrated that FABP3 knockout mice show decreased α-Synuclein oligomerization and neuronal degeneration of tyrosine hydroxylase (TH)-positive neurons in vivo. In this study, we newly investigated the importance of FABP3 in α-Synuclein uptake, 1-methyl-4-phenylpyridinium (MPP⁺)-induced axodendritic retraction, and mitochondrial dysfunction. To disclose the issues, we employed cultured mesencephalic neurons derived from wild type or FABP3^-/- C57BL6 mice and performed immunocytochemical analysis. We demonstrated that TH⁺ neurons from FABP3^+/+ mice take up α-Synuclein monomers while FABP3^-/- TH⁺ neurons do not. The formation of filamentous α-Synuclein inclusions following treatment with MPP⁺ was observed only in FABP3^+/+, and not in FABP3^-/- neurons. Notably, detailed morphological analysis revealed that FABP^-/- neurons did not exhibit MPP⁺-induced axodendritic retraction. Moreover, FABP3 was also critical for MPP⁺-induced reduction of mitochondrial activity and the production of reactive oxygen species. These data indicate that FABP3 is critical for α-Synuclein uptake in dopaminergic neurons, thereby preventing synucleinopathies, including Parkinson's disease.

Keywords: 1-methyl-4-phenylpyridinium (MPP+); Parkinson’s disease; fatty acid-binding protein 3; mitochondria; synucleinopathy; α-Synuclein.

Figure 1

Cultured primary dopaminergic neurons require fatty acid-binding protein 3 (FABP3) to take up α-Synuclein. (A) Representative images of TH⁺ mesencephalic neurons at days in vitro (DIV) 12 derived from wild type (WT) or FABP3^−/− C57BL6 mice. Neurons were exposed to 1 μM ATTO-550-labeled α-Synuclein monomer for 48 h and stained with antibody against tyrosine hydroxylase (TH, green). Scale bar: 10 μm. (B) Quantitative analysis of the ratio of ATTO-550-labeled α-Synuclein fluorescence intensity (FL) to TH immunoreactivity in the region of interest (ROI)-selected soma of individual TH⁺ neurons shown in A (white square 15 × 15 μm). **** p < 0.0001 in wild type (WT) versus FABP3^−/− (KO), n > 20. (C) Ratio of ATTO-550 fluorescence intensity to TH immunoreactivity in ROI-selected neuronal processes shown in A (white square 10 × 30 μm). **** p < 0.0001 in WT versus KO, n > 60. (D) The calculated ratio of ATTO-550 to TH in the individual terminal (C) was divided by the value in the soma (B) to represent the superiority on the α-Synuclein uptake in the axonal processes compared to the uptake in the soma. **** p < 0.0001 in WT versus KO, n > 20.

Figure 2

FABP3 is required for the formation of α-Synuclein inclusions in 1-methyl-4-phenylpyridinium (MPP⁺)-treated dopaminergic neurons. (A) Representative images of TH⁺ mesencephalic neurons at DIV 12 derived from wild type (WT) and FABP3^−/− C57BL6 mice. Neurons were exposed to 1 μM ATTO-550-labeled α-Synuclein monomer with or without 10 μM MPP⁺ for 48 h. The cells are stained with antibodies against TH (green) and FABP3 (blue). The dotted circles indicate α-Synuclein and FABP3-positive aggregates. Scale bar: 20 μm. (B) Confocal microscopy reveals ATTO-labelled α-Synuclein within the intracellular compartment of dopaminergic neurons. Image taken from an optical section approximately in the center of the z-axis of a dopaminergic neuron stained with anti-TH and anti-FABP3 antibodies. The red dotted circles indicate α-Synuclein and FABP3-positive aggregates. Scale bar: 10 μm. (C) Representative images of TH⁺ mesencephalic neurons either treated or not treated with MPP⁺. The images were acquired in photon counting mode using Leica TCS SP8 with a higher magnification (×63 objective). Scale bar: 10 μm. (D) Quantitative analysis of ATTO-550-labeled α-Synuclein monomer fluorescence intensity in ROI-selected soma of individual TH⁺ neurons shown in C (white square). **** p < 0.0001, ** p < 0.01, * p < 0.05 versus WT control; ^$$$$ p < 0.0001 versus WT MPP⁺; n > 60. (E) Percentage of ATTO-550-labeled α-Synuclein-positive inclusions (defined as dot-like signals 3 standard deviations (SD) above the background levels) in the total cell area [31]. **** p < 0.0001 versus WT control; ^$$$$ p < 0.0001 versus WT MPP⁺; n > 60. (F) Representative images of TH⁺ mesencephalic neurons in the same treatment conditions as the neurons in A. The cells are stained with antibodies against α-Synuclein filament (green) and TH (blue). Scale bar: 20 μm. (G) Representative images of a TH⁺ mesencephalic neuronal process stained with an antibody against TH (green). The magnified images of the dotted box are shown in the right, and the white arrowheads indicate Lewy neurite-like structures. Scale bar: 10 μm.

Figure 3

Representative dendritic morphology of TH⁺ dopaminergic neurons derived from wild type or FABP3^−/− mice. (A) Representative traced dendritic patterns of wild type (WT) or FABP3^−/− (KO) dopaminergic neurons either treated or not treated with 10 μM MPP⁺ for 48 h. (B) Sholl analysis of WT or FABP3 KO dopaminergic neurons either treated or not treated with MPP⁺. **** p < 0.0001, ** p < 0.01, * p < 0.05 versus WT control, n > 20. (C) Average neurite length of WT or FABP3^−/− dopaminergic neurons. **** p < 0.0001 versus WT control; ^$$$$ p < 0.0001 versus WT MPP⁺, n > 20.

Figure 4

FABP3 is involved in MPP⁺-induced reduction of mitochondrial activity and ROS production in dopaminergic neurons. (A) Representative images of JC-1-stained TH⁺ neurons. The white boxes indicate regions of interest (ROIs), which are magnified in the upper right in each image and quantified to measure the fluorescence intensity. Scale bar: 10 μm. (B) Quantitative analysis of Ex590 (red) and Ex529 (green) fluorescence intensity in JC-1-stained TH⁺ neurons. **** p < 0.0001 versus wild type (WT) control; ^$$$$ p < 0.0001 versus WT MPP⁺, n > 20. (C) Representative images of TH⁺ neurons (red) stained with anti-4HNE antibody (green). The white boxes show ROIs to measure 4-HNE fluorescence intensity. Scale bar: 10 μm. (D) Quantitative analysis of 4-HNE immunoreactivity in TH⁺ cells. **** p < 0.0001 versus WT control; ^$$$$ p < 0.0001 versus WT MPP⁺, n > 20.