CRISPR/Cas9 Editing of Glia Maturation Factor Regulates Mitochondrial Dynamics by Attenuation of the NRF2/HO-1 Dependent Ferritin Activation in Glial Cells

Abstract

Microglial cells are brain specific professional phagocytic immune cells that play a crucial role in the inflammation- mediated neurodegeneration especially in Parkinson's disease (PD) and Alzheimer's disease. Glia maturation factor (GMF) is a neuroinflammatory protein abundantly expressed in the brain. We have previously shown that GMF expression is significantly upregulated in the substantia nigra (SN) of PD brains. However, its possible role in PD progression is still not fully understood. The Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR Associated (Cas) protein9 (CRISPR/Cas9) system is a simple, rapid and often extremely efficient gene editing tool at desired loci, enabling complete gene knockout or homology directed repair. In this study, we examined the effect of GMF editing by using the CRISPR/Cas9 technique in BV2 microglial cells (hereafter referred to as BV2-G) on oxidative stress and nuclear factor erythroid 2-related factor 2 (NRF2)/Hemeoxygenase1 (HO-1)-dependent ferritin activation after treatment with (1-methyl-4-phenylpyridinium) MPP⁺. Knockout of GMF in BV2-G cells significantly attenuated oxidative stress via reduced ROS production and calcium flux. Furthermore, deficiency of GMF significantly reduced nuclear translocation of NRF2, which modulates HO-1 and ferritin activation, cyclooxygenase 2 (COX2) and nitric oxide synthase 2 (NOS2) expression in BV2 microglial cells. Lack of GMF significantly improved CD11b and CD68 positive microglial cells as compared with untreated cells. Our results also suggest that pharmacological and genetic intervention targeting GMF may represent a promising and a novel therapeutic strategy in controlling Parkinsonism by regulating microglial functions. Targeted regulation of GMF possibly mediates protein aggregation in microglial homeostasis associated with PD progression through regulation of iron metabolism by modulating NRF2-HO1 and ferritin expression.

Keywords: CRISPR/Cas9; Glia maturation factor; Microglial cells; Parkinson’s disease.

Fig. 1

Effect of GMF on MPP⁺ induced oxidative stress in BV2 microglial cells. The BV2 and BV2-G microglial cells were seeded (3×10⁶) in a 96-well plate. Then the cells were pre-incubated with DCFH-DA dye for 45 min and the cells were treated with MPP⁺ (0.1 mM) for 24 h. After the incubation period, cells were used to measure ROS intensity on a fluorescence microscope. Representative images show the toxic effect of MPP⁺ induced ROS intensity (green fluorescence; A). Bar graphs show the green fluorescence intensity significantly reduced in GMF deficient BV2-G cells as compared with BV2 microglial cells (B). Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. The p value less than <0.05 was considered as statistically significant in all the experiments; Values are given as mean ± SEM of three experiments in each group. p<0.001 and p<0.01 untreated controls vs MPP⁺-treated cells; p<0.05 MPP⁺-treated BV2 cells vs MPP⁺-treated BV2-G cells. Scale bar 100 µm. AU arbitrary unit.

Fig. 2

Effect of GMF on MPP⁺ induced apoptosis in BV2 microglial cells. The BV2 and BV2-G cells were seeded (3×10⁶) in a 96-well plate. The cells were treated with MPP⁺ (0.1 mM) for 24 h. After the incubation period, cells were stained with EtBr/AO solution (1:1 v/v) at a final concentration of 100 µg/ml for 5 min and finally imaged under fluorescence microscope. Images represents the toxic effect of MPP⁺ -induced apoptotic cell death (dark orange red; Fig 2A). Bar graphs show that apoptotic changes were significantly reduced in BV2-G microglial cells as compared with BV2 cells (B). Values are given as mean ± SEM of three experiments in each group. Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. p<0.001 untreated controls vs MPP⁺-treated cells; p<0.01 MPP⁺-treated BV2 cells vs MPP⁺-treated BV2-G cells. The p value less than <0.05 was considered as statistically significant in all the experiments; Scale bar 100 µm.

Fig. 3

Effect of GMF on MPP⁺ induced Ca²⁺ influx in BV2 microglial cells. The BV2 and BV2-G cells were seeded (3×10⁶) in a 96-well plate. Then the cells were pre-stained using Fura-2 QBT Calcium Assay kit according to the kit manufacturer’s instruction. The cells were treated MPP⁺ (0.1 mM) and plates were transferred to a FLEX Station3 multi-mode benchtop scanning fluorometer chamber (Molecular Devices). Representative results show the effect of MPP⁺ induced Ca²⁺ mobilization (flux) which is significantly reduced in BV2-G cells as compared with BV2 microglial cells. Values are given as mean ± SEM of three experiments in each group and represented as arbitrary unit.

Fig. 4

Effect of GMF on MPP⁺ induced NRF2 translocation in BV2 microglial cells. The BV2 and BV2-G cells were seeded in a T25 culture flask and cultured under standard conditions. Cells were incubated with MPP⁺ (0.1 mM) for 24 h. After the incubation period, cells were washed with PBS lysed and cytosolic and nuclear fractions separated. Aliquots were subjected to western blot analysis (A). Decreased nuclear and increased cytosolic expression level of NRF2 was found in BV2-G cells as compared with BV2 cells. Bar graphs shows the mean densitometry analysis of bands after normalizing with β-actin as a loading control of each group (B and C). Values are given as mean ± SEM of three experiments in each group. Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. p<0.001 untreated controls vs MPP⁺-treated cells; p<0.05 and p<0.001 MPP⁺-treated BV2 cells vs MPP⁺-treated BV2-G cells. The p value less than <0.05 was considered as statistically significant in all the experiments.

Fig. 5

Effect of GMF on MPP⁺ induced HO-1 and ferritin expression in BV2 microglial cells. The BV2 and BV2-G cells were seeded in a T25 culture flask and cultured under standard conditions. The cells were incubated with MPP⁺ (0.1 mM) for 24 h. After the incubation period, cells were washed with PBS and prepared for western blot analysis. Decreased HO-1 and ferritin expressions were found in BV2-G cells as compared with BV2 cells (A). Bar graphs show the mean densitometry analysis of bands after normalizing with β-actin as a loading control of each group (B and C). Values are given as mean ± SEM of three experiments in each group. Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. p<0.001 untreated controls vs MPP⁺-treated cells; p<0.05 MPP⁺-treated BV2 cells vs MPP⁺-treated BV2-G cells. The p value less than <0.05 was considered as statistically significant in all the experiments.

Fig. 6

Effect of GMF on MPP⁺ induced HO-1 and ferritin expression in BV2 microglial cells. The BV2 and BV2-G microglial cells were seeded on poly-D-lysine coated coverslips. The cells were incubated with MPP⁺ (0.1 mM) for 24 h. After the incubation period, cells were washed with PBS and processed for immunocytochemistry to detect HO-(A) and ferritin (D) co-localization along with NRF2 expression. Quantitatively decreased total positive area and total average intensity of HO-1 (B and C) and ferritin (E and F) along with NRF2 translocation to nuclear site were found in BV2-G cells as compared with BV2 microglial cells. Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. p<0.05 untreated controls vs MPP⁺-treated cells; p<0.05 MPP⁺-treated BV2 cells vs MPP⁺-treated BV2-G cells. The p value less than <0.05 was considered as statistically significant in all the experiments; Scale bar 100 µm; au arbitrary units

Fig. 7

Effect of GMF on MPP⁺ induced COX2 and NOS2 expression in BV2 microglial cells. The BV2 and BV2-G microglial cells were seeded in a T25 culture flask. The cells were incubated with MPP⁺ (0.1 mM) for 24 h. After the incubation period, cells were washed with PBS and cell lysates were prepared from these cells for western blot studies. Decreased expression of COX2 and NOS2 were found in BV2-G cells when compared with BV2 microglial cells (A). Bar graphs show the mean densitometry analysis of bands after normalizing with β-actin as a loading control of each group (B and C). Values are given as mean ± SEM of three experiments in each group. Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. p<0.001 and p<0.01 untreated controls vs MPP⁺-treated cells; p<0.01 and p<0.05 MPP⁺-treated BV2 cells vs MPP⁺-treated BV2-G cells. The p value less than <0.05 was considered as statistically significant in all the experiments.

Fig. 8

Effect of GMF on MPP⁺ induced activated microglial markers CD11b and CD68 expression in BV2 microglial cells. The BV2 and BV2-G microglial cells were seeded in T25 culture flask and poly-D-lysine coated coverslips. Then the cells were incubated with MPP⁺ (0.1 mM) for 24 h. After the incubation period, cells were washed with PBS and prepared for immunocytochemistry (A) and FACS analysis (B) of CD11b and CD68. Reduced expression levels of CD11b and CD68 were found in BV2-G cells when compared with BV2 microglial cells. FACS results shows the CD68-APC-A750-A (red fluorescent) versus CD11B-FITC-A (green fluorescent) derived fluorescence of BV2 and GMF deficient BV2-G cells in the plot panels (B). All data represent one of at least three separate experiments.