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Cited by 188Neuroinflammation and Neurodegeneration MechanismsParkinson's Disease Mechanisms and TreatmentsNeurological Disease Mechanisms and Treatments

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Microglial activation contributes to cognitive impairments in rotenone-induced mouse Parkinson’s disease model

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Zhang, Dongdong, Li, Sheng, Hou, Liyan, Jing, Lu, Ruan, Zhengzheng, Peng, Bingjie, Zhang, Xiaomeng, Hong, Jau-Shyong, Zhao, Jie, Wang, Qingshan·2021·Journal of Neuroinflammation
DOI 10.1186/s12974-020-02065-zPMC PMC7786472

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Abstract

BACKGROUND: Cognitive decline occurs frequently in Parkinson's disease (PD), which greatly decreases the quality of life of patients. However, the mechanisms remain to be investigated. Neuroinflammation mediated by overactivated microglia is a common pathological feature in multiple neurological disorders, including PD. This study is designed to explore the role of microglia in cognitive deficits by using a rotenone-induced mouse PD model. METHODS: To evaluate the role of microglia in rotenone-induced cognitive deficits, PLX3397, an inhibitor of colony-stimulating factor 1 receptor, and minocycline, a widely used antibiotic, were used to deplete or inactivate microglia, respectively. Cognitive performance of mice among groups was detected by Morris water maze, objective recognition, and passive avoidance tests. Neurodegeneration, synaptic loss, α-synuclein phosphorylation, glial activation, and apoptosis were determined by immunohistochemistry and Western blot or immunofluorescence staining. The gene expression of inflammatory factors and lipid peroxidation were further explored by using RT-PCR and ELISA kits, respectively. RESULTS: Rotenone dose-dependently induced cognitive deficits in mice by showing decreased performance of rotenone-treated mice in the novel objective recognition, passive avoidance, and Morris water maze compared with that of vehicle controls. Rotenone-induced cognitive decline was associated with neurodegeneration, synaptic loss, and Ser129-phosphorylation of α-synuclein and microglial activation in the hippocampal and cortical regions of mice. A time course experiment revealed that rotenone-induced microglial activation preceded neurodegeneration. Interestingly, microglial depletion by PLX3397 or inactivation by minocycline significantly reduced neuronal damage and α-synuclein pathology as well as improved cognitive performance in rotenone-injected mice. Mechanistically, PLX3397 and minocycline attenuated rotenone-induced astroglial activation and production of cytotoxic factors in mice. Reduced lipid peroxidation was also observed in mice treated with combined PLX3397 or minocycline and rotenonee compared with rotenone alone group. Finally, microglial depletion or inactivation was found to mitigate rotenone-induced neuronal apoptosis. CONCLUSIONS: Taken together, our findings suggested that microglial activation contributes to cognitive impairments in a rotenone-induced mouse PD model via neuroinflammation, oxidative stress, and apoptosis, providing novel insight into the immunopathogensis of cognitive deficits in PD.

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Fig. 1 Rotenone dose-dependently impairs the cognitive capacity of mice. a Experimental design. Mice were treated with different doses of rotenone. Three weeks after the initial injection, the Morris water maze (MWM) tests were performed, followed by novel objective recognition (NOR) and passive avoidance tests (PAT). After the behavior tests, mice were sacrificed and the brains were collected. For MWM test, different parameters, including escape latency (b), distance traveled (c), swimming speed (d), first platform crossing latency (e), number of platform crossings (f), and time percentage in target quadrant (g) were recorded. h For NOR test, recognition index was recorded. For PAT test, step-through latency (i) and error times in step-through (j) were recorded. n = 12; *p < 0.05, **p < 0.01
Fig. 1 Rotenone dose-dependently impairs the cognitive capacity of mice. a Experimental design. Mice were treated with different doses of rotenone. Three weeks after the initial injection, the Morris water maze (MWM) tests were performed, followed by novel objective recognition (NOR) and passive avoidance tests (PAT). After the behavior tests, mice were sacrificed and the brains were collected. For MWM test, different parameters, including escape latency (b), distance traveled (c), swimming speed (d), first platform crossing latency (e), number of platform crossings (f), and time percentage in target quadrant (g) were recorded. h For NOR test, recognition index was recorded. For PAT test, step-through latency (i) and error times in step-through (j) were recorded. n = 12; *p < 0.05, **p < 0.01CC BY · PubMed Central
Fig. 2 Rotenone dose-dependently induces neurodegeneration and α-synuclein Ser129 phosphorylation in mice. a The representative images of Neu-N and PSD95 immunostaining in mice were shown. b–e Quantitative analysis of Neu-N+ cell number and PSD-95 immunostaining density in the dentate gyrus (DG) and cortex of mice. n = 5–6. f The representative blots of Neu-N and PSD-95 detected by Western blot were shown. g, h Quantitative analysis of Neu-N and PSD95 blots. n = 6. i The representative images of Ser129-phosphorylated α-synuclein immunostaining in the hippocampus and cortex of mice were shown. j Quantitative analysis of the density of Ser129-phosphorylated α-synuclein immunostaining. n = 4. *p < 0.05, **p < 0.01; Scale bar = 200 and 50 μm in A and I, respectively
Fig. 2 Rotenone dose-dependently induces neurodegeneration and α-synuclein Ser129 phosphorylation in mice. a The representative images of Neu-N and PSD95 immunostaining in mice were shown. b–e Quantitative analysis of Neu-N+ cell number and PSD-95 immunostaining density in the dentate gyrus (DG) and cortex of mice. n = 5–6. f The representative blots of Neu-N and PSD-95 detected by Western blot were shown. g, h Quantitative analysis of Neu-N and PSD95 blots. n = 6. i The representative images of Ser129-phosphorylated α-synuclein immunostaining in the hippocampus and cortex of mice were shown. j Quantitative analysis of the density of Ser129-phosphorylated α-synuclein immunostaining. n = 4. *p < 0.05, **p < 0.01; Scale bar = 200 and 50 μm in A and I, respectivelyCC BY · PubMed Central
Fig. 3 Rotenone-induced microglial activation precedes neurodegeneration in mice. a The representative images of Iba-1 immunostaining were shown. b, c Quantitative analysis of Iba-1 immunostaining density. n = 5–6. d Experimental design. Mice were administered with 1.5 mg/kg rotenone. After 1, 2, and 3 weeks of initial exposure, Iba-1, Neu-N, and PSD95 were immunostained in the hippocampus and cortex. e The representative images of Iba-1, Neu-N, and PSD95 immunostaining were shown. e, f Quantitative analysis of the densities of Iba-1 and PSD95 immunostaining and the number of Neu-N+ cells in mice. n = 4. *p < 0.05, **p < 0.01; Scale bar = 50 μm
Fig. 3 Rotenone-induced microglial activation precedes neurodegeneration in mice. a The representative images of Iba-1 immunostaining were shown. b, c Quantitative analysis of Iba-1 immunostaining density. n = 5–6. d Experimental design. Mice were administered with 1.5 mg/kg rotenone. After 1, 2, and 3 weeks of initial exposure, Iba-1, Neu-N, and PSD95 were immunostained in the hippocampus and cortex. e The representative images of Iba-1, Neu-N, and PSD95 immunostaining were shown. e, f Quantitative analysis of the densities of Iba-1 and PSD95 immunostaining and the number of Neu-N+ cells in mice. n = 4. *p < 0.05, **p < 0.01; Scale bar = 50 μmCC BY · PubMed Central
Fig. 4 PLX3397 and minocycline attenuate rotenone-induced cognitive deficits in mice. a Experimental design. Mice were preadministered with PLX3397 daily. Seven days later, mice received PLX3397 1 h after the rotenone injection every other day until the end of the experiment. Minocycline was administered (i.p.) to mice 2 days before rotenone and continued for consecutive 3 weeks. Morris water maze (MWM) tests were performed and followed by novel objective recognition (NOR) and passive avoidance tests (PAT) in rotenone-treated mice with or without PLX3397 and minocycline. a Escape latency, b distance traveled, c swimming speed, d first platform crossing latency, e number of platform crossings, and f time percentage in target quadrant were recorded. g–i Novel objective recognition and passive avoidance test were performed in rotenone-treated mice with or without PLX3397 and minocycline. g Recognition index, h step-through latency, and i error times in step-through were recorded. n = 11–12. *p < 0.05, **p < 0.01
Fig. 4 PLX3397 and minocycline attenuate rotenone-induced cognitive deficits in mice. a Experimental design. Mice were preadministered with PLX3397 daily. Seven days later, mice received PLX3397 1 h after the rotenone injection every other day until the end of the experiment. Minocycline was administered (i.p.) to mice 2 days before rotenone and continued for consecutive 3 weeks. Morris water maze (MWM) tests were performed and followed by novel objective recognition (NOR) and passive avoidance tests (PAT) in rotenone-treated mice with or without PLX3397 and minocycline. a Escape latency, b distance traveled, c swimming speed, d first platform crossing latency, e number of platform crossings, and f time percentage in target quadrant were recorded. g–i Novel objective recognition and passive avoidance test were performed in rotenone-treated mice with or without PLX3397 and minocycline. g Recognition index, h step-through latency, and i error times in step-through were recorded. n = 11–12. *p < 0.05, **p < 0.01CC BY · PubMed Central
Fig. 5 PLX3397 and minocycline attenuate rotenone-induced neuronal damage in mice. a The representative images of Neu-N and PSD95 immunostaining in rotenone-treated mice with or without PLX3397 and minocycline were shown. High magnification images of PSD95 staining were shown in Supplementary Fig. 4. b–e Quantitative analysis of Neu-N+ cell number and PSD-95 immunostaining density in the dentate gyrus (DG) and cortex of mice. n = 5–6. f The representative blots of Neu-N and PSD-95 detected by Western blot were shown. g, h Quantitative analysis of Neu-N and PSD95 blots. n = 4. i The representative images of Ser129-phosphorylated α-synuclein immunostaining in the hippocampus and cortex of mice were shown. j Quantitative analysis of the density of Ser129-phosphorylated α-synuclein immunostaining. n = 4. *p < 0.05, **p < 0.01; Scale bar = 200 and 50 μm in a and i, respectively
Fig. 5 PLX3397 and minocycline attenuate rotenone-induced neuronal damage in mice. a The representative images of Neu-N and PSD95 immunostaining in rotenone-treated mice with or without PLX3397 and minocycline were shown. High magnification images of PSD95 staining were shown in Supplementary Fig. 4. b–e Quantitative analysis of Neu-N+ cell number and PSD-95 immunostaining density in the dentate gyrus (DG) and cortex of mice. n = 5–6. f The representative blots of Neu-N and PSD-95 detected by Western blot were shown. g, h Quantitative analysis of Neu-N and PSD95 blots. n = 4. i The representative images of Ser129-phosphorylated α-synuclein immunostaining in the hippocampus and cortex of mice were shown. j Quantitative analysis of the density of Ser129-phosphorylated α-synuclein immunostaining. n = 4. *p < 0.05, **p < 0.01; Scale bar = 200 and 50 μm in a and i, respectivelyCC BY · PubMed Central
Fig. 6 PLX3397 and minocycline attenuate rotenone-induced astroglial activation and gene expression of proinflammatory factors in mice. a The representative images of astroglial marker, GFAP immunostaining were shown. b, c Quantitative analysis of GFAP immunostaining density. d, e The gene expression levels of iNOS, TNFα, and IL-1β in the hippocampus (d) and cortex (e) of mice were detected. n = 5–6. *p < 0.05, **p < 0.01; Scale bar = 200 μm
Fig. 6 PLX3397 and minocycline attenuate rotenone-induced astroglial activation and gene expression of proinflammatory factors in mice. a The representative images of astroglial marker, GFAP immunostaining were shown. b, c Quantitative analysis of GFAP immunostaining density. d, e The gene expression levels of iNOS, TNFα, and IL-1β in the hippocampus (d) and cortex (e) of mice were detected. n = 5–6. *p < 0.05, **p < 0.01; Scale bar = 200 μmCC BY · PubMed Central
Fig. 7 PLX3397 and minocycline attenuate rotenone-induced oxidative stress in mice. The MDA (a, c) and GSH (b, d) contents in rotenone-treated mice with or without PLX3397 and minocycline were determined. n = 6. *p < 0.05, **p < 0.01
Fig. 7 PLX3397 and minocycline attenuate rotenone-induced oxidative stress in mice. The MDA (a, c) and GSH (b, d) contents in rotenone-treated mice with or without PLX3397 and minocycline were determined. n = 6. *p < 0.05, **p < 0.01CC BY · PubMed Central
Fig. 8 PLX3397 and minocycline attenuate rotenone-induced apoptosis in mice. a TUNEL staining was performed in rotenone-treated mice with or without PLX3397 or minocycline. b Quantitative analysis of TUNEL+ cells. c The representative blots of Bcl-XL, Bax, and active caspase-3 detected by Western blot were shown. d, e Quantitative analysis of Bcl-xL, Bax, and active caspase-3 blots. n = 4. *p < 0.05, **p < 0.01; Scale bar = 200 μm
Fig. 8 PLX3397 and minocycline attenuate rotenone-induced apoptosis in mice. a TUNEL staining was performed in rotenone-treated mice with or without PLX3397 or minocycline. b Quantitative analysis of TUNEL+ cells. c The representative blots of Bcl-XL, Bax, and active caspase-3 detected by Western blot were shown. d, e Quantitative analysis of Bcl-xL, Bax, and active caspase-3 blots. n = 4. *p < 0.05, **p < 0.01; Scale bar = 200 μmCC BY · PubMed Central

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Authors
Zhang, D., Li, S., Hou, L., Jing, L., Ruan, Z., Peng, B., Zhang, X., Hong, J.-S., Zhao, J., & Wang, Q.
Affiliations
  • Dalian Medical University (CN)
  • National Institutes of Health (US)
Corresponding author
  • Zhao, Jie
  • Wang, Qingshan
ORCID
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2021-01-05
Volume
18
Issue
1
Pages
4-4
ISSN
1742-2094 (ISSN-L)
DOI
10.1186/s12974-020-02065-z
PMC
PMC7786472
OpenAlex
W3111358404
Cited by
187
References
70
Citations by year
2026: 18 · 2025: 40 · 2024: 65 · 2023: 27 · 2022: 24 · 2021: 13
FWCI
12.91
Type
journal article
Language
en
Update notices
correction (2025-05-24)
Open access
Licence
Locations
  • Journal of Neuroinflammation (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (81973087)
  • Natural Science Foundation of Liaoning Province (2019-MS-077)
  • LiaoNing Revitalization Talents Program (XLYC1907026)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Clinical Neurology › Parkinson's Disease Mechanisms and Treatments
  • Life Sciences › Neuroscience › Neurology › Neurological Disease Mechanisms and Treatments
Keywords
Parkinson's disease, cognitive impairment, microglial activation, neuroinflammation, rotenone, PLX3397, minocycline, microglial depletion, alpha-synuclein phosphorylation, neurodegeneration, synaptic loss, astroglial activation, lipid peroxidation, oxidative stress, neuronal apoptosis, hippocampus, immunopathogenesis
MeSH terms
Aminopyridines, Animals, Insecticides, Male, Mice, Inbred C57BL, Minocycline, Pyrroles, Rotenone, Microglia, Parkinsonian Disorders, Mice, Cognitive Dysfunction

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