Molecular Association of Glia Maturation Factor with the Autophagic Machinery in Rat Dopaminergic Neurons: a Role for Endoplasmic Reticulum Stress and MAPK Activation

Abstract

Parkinson's disease (PD) is one of the several neurodegenerative diseases where accumulation of aggregated proteins like α-synuclein occurs. Dysfunction in autophagy leading to this protein build-up and subsequent dopaminergic neurodegeneration may be one of the causes of PD. The mechanisms that impair autophagy remain poorly understood. 1-Methyl-4-phenylpiridium ion (MPP⁺) is a neurotoxin that induces experimental PD in vitro. Our studies have shown that glia maturation factor (GMF), a brain-localized inflammatory protein, induces dopaminergic neurodegeneration in PD and that suppression of GMF prevents MPP⁺-induced loss of dopaminergic neurons. In the present study, we demonstrate a molecular action of GMF on the autophagic machinery resulting in dopaminergic neuronal loss and propose GMF-mediated autophagic dysfunction as one of the contributing factors in PD progression. Using dopaminergic N27 neurons, primary neurons from wild type (WT), and GMF-deficient (GMF-KO) mice, we show that GMF and MPP⁺ enhanced expression of MAPKs increased the mammalian target of rapamycin (mTOR) activation and endoplasmic reticulum stress markers such as phospho-eukaryotic translation initiation factor 2 alpha kinase 3 (p-PERK) and inositol-requiring enzyme 1α (IRE1α). Further, GMF and MPP⁺ reduced Beclin 1, focal adhesion kinase (FAK) family-interacting protein of 200 kD (FIP200), and autophagy-related proteins (ATGs) 3, 5, 7, 16L, and 12. The combined results demonstrate that GMF affects autophagy through autophagosome formation with significantly reduced lysosomal-associated membrane protein 1/2, and the number of autophagic acidic vesicles. Using primary neurons, we show that MPP⁺ treatment leads to differential expression and localization of p62/sequestosome and in GMF-KO neurons, there was a marked increase in p62 staining implying autophagy deficiency with very little co-localization of α-synuclein and p62 as compared with WT neurons. Collectively, this study provides a bidirectional role for GMF in executing dopaminergic neuronal death mediated by autophagy that is relevant to PD.

Keywords: Autophagy dysfunction; Glia maturation factor; Parkinson’s disease; Protein aggregation.

Fig. 1

GMF activates p38 and ERK1/2 MAPKs expression in dopaminergic N27 cells. N27 cells were seeded in T25 cell culture flasks and incubated with GMF (100 ng/ml) and MPP⁺ (300 μm) for 24 h under standard conditions. Then the cells were washed with PBS and cell lysates were prepared for western blot, using specific antibodies to p-p38, p-ERK1/2, p38 and ERK1/2. GMF treatment significantly increased p-p38 and p-ERK1/2 expression as compared with control cells (A and C). N27 cells simultaneously incubated with both GMF and MPP⁺ significantly increased p-p38 and p-ERK1/2 expression as compared with other group. Western blot results were quantified by ChemiDoc-It² imaging system analysis software, and the values are expressed as arbitrary units and given as mean ± SEM (n=4). Bar graphs show the effect of GMF exposure on the relative intensity compared to the control (B and D). *p< 0.05 compared to control. Statistical significance was assessed with one-way ANOVA followed by Tukey’s Kramer procedure using GraphPad prism-7 software.

Fig. 2

GMF increases target of rapamycin (mTOR) expression in dopaminergic N27 cells. N27 cells were seeded in T25 cell culture flask and incubated with GMF (100 ng/ml), MPP⁺ (300 μm), rapamycin (200 μM) and cyclosporine A (100 nM) for 24 h under standard conditions. Then the cells were washed with PBS and cell lysates were prepared for western blot analysis, using specific antibodies to total and p-mTOR, expression. GMF, MPP⁺ and cyclosporine A treatment significantly increased expression of p-mTOR when compared with control cells (A). Western blot results were quantified by ImageJ, and the values are expressed as percentage of control and given as mean ± SEM (n=4). Bar graphs show the effect of GMF, MPP⁺, rapamycin and cyclosporine A exposure on the relative intensity compared to the control (B). *p< 0.001 and *p< 0.05 compared to control and GMF. *p< 0.001 compared with MPP⁺ and GMF+MPP⁺. Statistical significance was assessed with one-way ANOVA followed by Tukey’s Kramer procedure and unpaired t-test was performed when comparing between groups by using GraphPad prism-7 software.

Fig. 3

Effect of GMF on FIP200 (ULK interacting protein) and Beclin 1 expression in dopaminergic N27 cells. N27 cells were seeded in T25 cell culture flask and incubated with GMF (100 ng/ml) and MPP⁺ (300 μm) for 24 h under standard conditions. After the incubation period, cells were washed with PBS and cell lysates were prepared from these cells for western blot using specific antibodies to FIP200, then stripped and reprobed for β-actin expression. The concentrations of Beclin 1 were determined by ELISA assay, using cell lysates collected from GMF and MPP⁺ treated cells. GMF and MPP⁺ treatment significantly reduced expression of FIP200 (A) and Beclin 1 (C) expression as compared with control cells. N27 cells simultaneously treated with both GMF and MPP⁺ significantly reduced FIP200 and Beclin 1 expression as compared with other group. Bar graphs showing the effect of GMF and MPP⁺ exposure on the percentage of control (C). Western blot results were quantified by ChemiDoc-It2 imaging system analysis software, and the values are given as mean ± SEM (n=4). Bar graphs show the effect of GMF exposure on the relative intensity to the control (B). *p< 0.05 compared to control, GMF, and MPP⁺. *p< 0.001 compared with MPP⁺ and GMF+MPP⁺. Statistical significance was assessed with one-way ANOVA followed by Tukey’s Kramer procedure and unpaired t-test was performed when comparing between groups by using GraphPad prism-7 software.

Fig. 4

GMF reduces ATG protein complexes expression in dopaminergic N27 cells. N27 cells were seeded in T25 cell culture flask and incubated with GMF (100 ng/ml) and MPP⁺ (300 μm) for 24 h under standard conditions. After the incubation period, cells were washed with PBS, cell lysates were prepared from these cells, and the concentrations of ATG protein complexes were determined by ELISA assay, using specific antibodies to ATG3, ATG5, ATG7, ATG16L and ATG12. GMF and MPP⁺ treatment significantly reduced expression of ATG protein complexes (A–E) as seen by ELISA results and compared with control cells. N27 cells incubated with both GMF and MPP⁺ significantly reduced expression of these proteins as compared with other group. Bar graphs show the effect of GMF and MPP⁺ exposure as a percentage of control (A–E) and the values are expressed as arbitrary units and given as mean ± SEM of four experiments in each group. *p< 0.05 compared to control. Statistical significance was assessed with one-way ANOVA followed by Tukey’s Kramer procedure using GraphPad prism-7 software.

Fig. 5

GMF increases ER stress markers expression in dopaminergic N27 cells. N27 cells were seeded and allowed to grow to confluency and incubated with GMF (100 ng/ml) and MPP⁺ (300 μm) for 24 h under standard conditions. After the incubation period, cells were washed with PBS and these cells prepared for western blot and immunofluorescence study, using specific antibodies such as p-PERK and IRE1α. GMF and MPP⁺ treatment significantly increased expression of p-PERK and IRE1α expression as compared with control cells (A, B, C and D). N27 cells treated simultaneously with both GMF and MPP⁺ significantly increased p-PERK and IRE1α expression as compared with other groups. Representative immunocytochemical images show the expression of IRE1α is reduced in the cytoplasmic region and also IRE1α translocates to the nuclei in the cells that were treated with GMF and/or MPP⁺ (Fig. 5E). Bar graphs show the effect of GMF and MPP⁺ exposure as a percentage of control (B and D). Western blot bands were quantified by ChemiDoc-It2 imaging system analysis software and the values are expressed as mean ± SEM (n=3). *p< 0.05 compared to control. Statistical significance was assessed with one-way ANOVA followed by Tukey’s Kramer procedure using GraphPad prism-7 software.

Fig. 6

GMF treatment increases LC3 and decreases LAMP1 immunocytochemical expression in rat dopaminergic N27 cells. N27 cells were incubated in the presence or absence of GMF (100 ng/ml) or MPP⁺ and immunostained for LC3 (green fluorescence) and LAMP1 (red fluorescence). Representative images show that exposure of N27 cells to GMF and MPP⁺ qualitatively increased LC3 and reduced LAMP1 expression as compared to control cells. Incubation of N27 cells with both GMF and MPP⁺ simultaneously qualitatively increased LC3 and reduced LAMP1 expression as compared with other group. Images were taken using Leica TCP SP8 laser scanning confocal microscope with a 405-nm diode laser and tunable super continuum white light laser using 63X oil immersion objective. Scale bar =100μm.

Fig. 7

GMF reduces lysosomal associated membrane proteins 1 and 2 (LAMP1 and 2) expression in dopaminergic N27 cells. N27 cells were seeded in T25 cell culture flask and incubated with GMF (100 ng/ml) and MPP⁺ (300 μm) for 24 h under standard conditions. After the incubation period, cells were washed with PBS and cell lysates were prepared from these cells for western blot analysis, using specific antibodies for LAMP1 and LAMP2. GMF and MPP⁺ significantly reduced the expression of LAMP1 and LAMP2 (indicated with arrow head) as compared with control cells. Incubation of N27 cells with both GMF and MPP⁺ simultaneously significantly reduced LAMP1 and LAMP 2 expression as compared with other group. Western blot bands were quantified by ChemiDoc-It2 imaging system analysis software and the values are expressed as mean ± SEM (n=3). *p< 0.05, *p < 0.001 and *p < 0.01 compared to control, GMF, and MPP⁺. *p< 0.001 compared with MPP⁺ and GMF+MPP⁺. Statistical significance was assessed with one-way ANOVA followed by Tukey’s Kramer procedure and unpaired t-test was performed when comparing between groups by using GraphPad prism-7 software.

Fig. 8

GMF decreases autophagic acidic vesicles or autophagosomes in dopaminergic N27 cells. N27 cells were incubated in the presence or absence of GMF (100 ng/ml) or MPP⁺ under standard condition. After incubation period cells were washed twice with PBS and stained with 10 μg/ml of acridine orange (AO) for autophagy lysosomal acidic vesicle (red fluorescence intensity) quantification. Representative images show that exposure of N27 cells to GMF significantly decreased the red fluorescence labelling intensity of AO with acidic vesicles correlates number of autophagy acidic vesicles when compared to control cells. Incubation of N27 cells with both GMF and MPP⁺ simultaneously significantly decreased autophagy acidic vesicles by reducing red fluorescents intensity as compared with other group (A). Images were taken using Leica TCP SP8 laser scanning confocal microscope with a 405-nm diode laser and tunable super continuum white light laser using 63X oil immersion objective. Scale bar = 100 μm. Autophagic acidic vesicle were quantified in the whole field by ImageJ, and the values are expressed as mean ± SEM (n=4). Bar graphs show the AO red fluorescence intensity and correlates with the number of acidic vesicles compared to the control (B). *p< 0.05 compared to control. Statistical significance was assessed with one-way ANOVA followed by Tukey’s Kramer procedure using GraphPad prism-7 software.

Fig. 9

GMF affects intracellular lysosomal morphology as detected by LysoTracker Red DND-99 fluorescent staining in dopaminergic N27 cells. N27 cells were seeded (3×10⁶) in 6 well plate with coverslip and incubated with GMF (100 ng/ml) and/or MPP⁺ (300 μm) for 24 h under standard conditions. After incubation period, the cells were washed with PBS. 1 mM of LysoTracker Red DND-99 (red fluorescence) added and coverslip mounted in a Vectashield [with DAPI for nuclear staining (blue florescence)] mounting media and finally visualized under Leica Confocal system. Representative confocal images show that GMF and MPP⁺ affects the cytoplasmic lysosomal mass compared with control cells (n=3). GMF and MPP⁺ qualitatively reduced the LysoTracker Red DND-99 fluorescent stained lysosomal mass as compared with control cells. Images were taken using Leica TCP SP8 laser scanning confocal microscope with a 405-nm diode laser and tunable super continuum white light laser using 63X oil immersion objective. Scale bar = 100 μm.

Fig. 10

GMF presence decreases p62/SQSTM immunocytochemical expression in murine primary neuronal cells. Primary WT and GMF-KO mouse neurons were incubated in the presence of MPP⁺ (20 μM) under standard condition. After incubation period, cells were immunostained for p62/SQSTM (green fluorescence) and counter stained with VectaShield DAPI mounting media for nuclear staining (blue fluorescence). Representative images (n=4) show that exposure of primary WT neurons to MPP⁺ qualitatively reduced p62/SQSTM expression (green color) as compared with control cells. GMF-KO primary neurons treated with MPP⁺ shows qualitatively increased p62/SQSTM expression as compared with WT primary neurons. Images were taken using Leica TCP SP8 laser scanning confocal microscope with a 405-nm diode laser and tunable super continuum white light laser using 63X oil immersion objective. Scale bar = 100 μm.

Fig. 11

GMF presence increases α-synuclein and downregulates p62/SQSTM immunocytochemical expression in mouse primary neurons. WT and GMF-KO mouse primary neurons were incubated in the presence of MPP⁺ (20 μM) under standard condition. After incubation period, cells were immunostained for α-synuclein (red fluorescence), p62/SQSTM (green fluorescence) and mounted with Vectashield [with DAPI for nuclear staining (blue fluorescent)]. Representative images show that exposure of WT primary neurons to MPP⁺ significantly increased α-synuclein and decreased p62/SQSTM expression as compared with control neurons. GMF-KO primary neurons treated with MPP⁺ shows increased p62/SQSTM and reduced α-synuclein expression as compared with WT primary neurons (n=3). Images were taken using Leica TCP SP8 laser scanning confocal microscope with a 405-nm diode laser and tunable super continuum white light laser using 63X oil immersion objective. Scale bar = 100 μm.