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. 2018 Dec 21:7:35.
doi: 10.1186/s40035-018-0138-4. eCollection 2018.

Dynamic changes of CX3CL1/CX3CR1 axis during microglial activation and motor neuron loss in the spinal cord of ALS mouse model

Jingjing Zhang  1   2   3 , Yufei Liu  1   2 , Xinyao Liu  1   2 , Song Li  1   2 , Cheng Cheng  1   2 , Sheng Chen  4 , Weidong Le  1   2
Affiliations

Dynamic changes of CX3CL1/CX3CR1 axis during microglial activation and motor neuron loss in the spinal cord of ALS mouse model

Jingjing Zhang et al. Transl Neurodegener. .

Abstract

Background: Neuron-microglia communication plays a crucial role in the motor neurons (MNs) death in amyotrophic lateral sclerosis (ALS). Neurons can express chemokine (C-X3-C motif) ligand 1 (CX3CL1), which mediates microglial activation via interacting with its sole receptor CX3CR1 in microglia. In the present study, we aimed to investigate the dynamic changes of CX3CL1/CX3CR1 axis during microglial activation and MNs loss in SOD1G93A mouse model of ALS.

Methods: qPCR, western blot and immunofluorescent staining were used to examine the mRNA and protein levels and localization of CX3CL1/CX3CR1 in the anterior horn region of spinal cord in both SOD1G93A mice and their age-matched wild type (WT) littermates at 40, 60, 90 and 120 days of age. The M1/M2 microglial activation in the spinal cord tissues of SOD1G93A mice and WT mice were evaluated by immunofluorescent staining of M1/M2 markers and further confirmed by qPCR analysis of M1/M2-related cytokines.

Results: The immunofluorescent staining revealed that CX3CL1 was predominately expressed in MNs, while CX3CR1 was highly expressed in microglia in the anterior horn region of spinal cord. Compared with age-matched WT mice, CX3CL1 mRNA level was elevated at 40 days but decreased at 90 and 120 days in the anterior horn region of spinal cords in ALS mice. Consistently, CX3CR1 mRNA level was increased at 90 and 120 days. Western blot assay further confirmed the dynamic changes of CX3CL1/CX3CR1 axis in ALS mice. Additionally, the levels of M1/M2 markers of microglia and their related cytokines in the anterior horn region of spinal cord in ALS mice were increased at 90 and 120 days. Moreover, while M1-related cytokines in ALS mice were persistently increased at 120 days, the upregulated M2-related cytokines started to decline at 120 days, suggesting an altered microglial activation.

Conclusions: Our data revealed the dynamic changes of CX3CL1/CX3CR1 axis and an imbalanced M1/M2 microglial activation during ALS pathological progression. These findings may help identify potential molecular targets for ALS therapy.

Keywords: ALS; CX3CL1/CX3CR1 axis; Microglial activation; SOD1G93A mice.

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Conflict of interest statement

All experimental procedures on animals in this study were conducted in accordance with the laboratory animals care guidelines and approved by institutional animal care committee at Dalian Medical University.Not applicable.The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
CX3CL1 were mainly located in neurons and decreased during MNs loss in TG mice. a Double immunofluorescent staining of CX3CL1 (green) and NeuN (red) in the anterior horn of lumber spinal cord of WT and TG mice at the age of 40, 90 and 120 days. Scale bar = 50 μm. b Integrated density of CX3CL1 staining. The values are expressed as mean ± SD, *p < 0.05 versus age-matched WT mice, ** p < 0.001 versus age-matched WT mice, n = 5 in each group
Fig. 2
Fig. 2
The mRNA and protein levels of CX3CL1 in the spinal cord anterior horns. a qPCR analysis of CX3CL1 mRNA level. b Western blot analysis of CX3CL1 protein level. c Quantitative analysis the CX3CL1 Protein level. The values are expressed as mean ± SD, *p < 0.05 versus age-matched WT mice, ** p < 0.001 versus age-matched WT mice, n = 5 in each group
Fig. 3
Fig. 3
CX3CR1 was mainly located in microglia and was increased in the TG mice at the age of 90 and 120 days. a Double immunofluorescent staining of CX3CR1 (green) and Iba1(red) in the anterior horn of lumber spinal cord of WT and TG mice at the age of 40, 90 and 120 days. Scale bar = 50 μm. b Number of CX3CR1+/Iba1+ microglia per mm2. The values are expressed as mean ± SD, *p < 0.05 versus age-matched WT mice, ** p < 0.001 versus age-matched WT mice, n = 5 in each group
Fig. 4
Fig. 4
The mRNA and protein levels of CX3CR1 in the spinal cord anterior horns. a qPCR analysis of CX3CR1 mRNA level. b Western blot analysis of CX3CR1 protein level. c Quantitative analysis the CX3CR1 Protein level. The values are expressed as mean ± SD, *p < 0.05 versus age-matched WT mice, ** p < 0.001 versus age-matched WT mice, n = 5 in each group
Fig. 5
Fig. 5
The changes of M1 microglia in the TG mice at different time points. a Double immunofluorescent staining of CD86 (red) and Iba1 (green) at different time points, Sale bar = 50 μm. b Quantitative analysis of the M1 microglia per mm2 at different time points. The values are expressed as mean ± SD, *p < 0.05 versus age-matched WT mice, ** p < 0.001 versus age-matched WT mice, n = 5 in each group
Fig. 6
Fig. 6
The changes of M2 microglial phenotypes in TG mice at different time points. a Double immunofluorescent staining of Arg1 (green) and Iba1 (red) at different time points, Sale bar = 50 μm. b Quantitative analysis of the M2 microglia per mm2 at different time points. The values are expressed as mean ± SD, *p < 0.05 versus age-matched WT mice, ** p < 0.001 versus age-matched WT mice, n = 5 in each group
Fig. 7
Fig. 7
The mRNA levels of M1/M2 microglial markers in the TG mice at different time points. a, b The mRNA levels of M1 markers (INOS, CD86). c, d The mRNA levels of M2 markers (Arg1, CD206). The values are expressed as mean ± SD, *p < 0.05 versus age-matched WT mice, ** p < 0.001 versus age-matched WT mice, n = 5 in each group
Fig. 8
Fig. 8
The changes of M1/M2 microglia related cytokines mRNA levels at different time points. a, b mRNA levels of two cytokines related to M1 microglia (IL-1β and TNF-α). c mRNA levels of IL-10 related to M2 microglia. The values are expressed as mean ± SD, *p < 0.05 versus age-matched WT mice, ** p < 0.001 versus age-matched WT mice, n = 5 in each group
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