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Cited by 150Alzheimer's disease research and treatmentsNeuroinflammation and Neurodegeneration MechanismsCerebrospinal fluid and hydrocephalus

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Microglia prevent beta-amyloid plaque formation in the early stage of an Alzheimer’s disease mouse model with suppression of glymphatic clearance

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Feng, Weixi, Zhang, Yanli, Wang, Ze, Xu, Hanrong, Wu, Ting, Marshall, Charles, Gao, Junying, Xiao, Ming·2020·Alzheimer's Research & Therapy
DOI 10.1186/s13195-020-00688-1PMC PMC7532614

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Abstract

Background: Soluble beta-amyloid (Aβ) can be cleared from the brain through various mechanisms including enzymatic degradation, glial cell phagocytosis, transport across the blood-brain barrier, and glymphatic clearance. However, the relative contribution of each clearance system and their compensatory effects in delaying the pathological process of Alzheimer’s disease (AD) are currently unknown. Methods: Fluorescent trace, immunofluorescence, and Western blot analyses were performed to compare glymphatic clearance ability and Aβ accumulation among 3-month-old APP695/PS1-dE9 transgenic (APP/PS1) mice, wild-type mice, aquaporin 4 knock out (AQP4 −/− ) mice, and AQP4 −/− /APP/PS1 mice. The consequence of selectively eliminating microglial cells, or downregulating apolipoprotein E (apoE) expression, on Aβ burden, was also investigated in the frontal cortex of AQP4 −/− /APP/PS1 mice and APP/PS1 mice. Results: AQP4 deletion in APP/PS1 mice significantly exaggerated glymphatic clearance dysfunction, and intraneuronal accumulation of Aβ and apoE, although it did not lead to Aβ plaque deposition. Notably, microglia, but not astrocytes, increased activation and phagocytosis of Aβ in the cerebral cortex of AQP4 −/− /APP/PS1 mice, compared with APP/PS1 mice. Selectively eliminating microglia in the frontal cortex via local injection of clodronate liposomes resulted in deposition of Aβ plaques in AQP4 −/− /APP/PS1 mice, but not APP/PS1 mice. Moreover, knockdown of apoE reduced intraneuronal Aβ levels in both APP/PS1 mice and AQP4 −/− /APP/PS1 mice, indicating an inhibitory effect of apoE on Aβ clearance. Conclusion: The above results suggest that the glymphatic system mediated Aβ and apoE clearance and microglia mediated Aβ degradation synergistically prevent Aβ plague formation in the early stages of the AD mouse model. Protecting one or both of them might be beneficial to delaying the onset of AD.

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Figures

Fig. 1 AQP4 deletion aggravates decreases of florescent tracer influx into, and efflux out of, the brain of 3-month-old APP/PS1 mice. a, b Schematic images for intracisternal injection and intrastriatal injection of TR-d3. c Representative images of serial coronal brain sections from 1.0 mm anterior to 1.8 mm posterior to bregma (upper panel) and high magnification micrographs of the sections at the level of 0.5 mm anterior to bregma (low panel) showing TR-d3 penetration into the brain parenchyma at 30 min after intracisternal injection. d High magnification micrographs of the cortex showing TR-d3 penetration into the perivascular space (star) and adjacent brain parenchyma (dotted line) at 30 min after intracisternal injection. e Representative images of serial coronal brain sections from 1.0 mm anterior to 1.8 mm posterior to bregma (upper panel) and high magnification micrographs of the sections at the level of 0.2 mm anterior to bregma (low panel) showing residue of TR-d3 within the striatum at 1 h after intracisternal injection. f Low (upper panel) and high (low panel) magnification micrographs of sections of the dcLNs showing TR-d3 distribution at 1 h after intracisternal injection. g Quantification of intracisternally injected TR-d3 area fraction in the brain. h Quantification of the mean fluorescence intensity of TR-d3 in the cerebral cortex after intracisternal injection. i Quantification of intrastriatally injected TR-d3 area in the brain. j Quantification of intrastriatal injected TR-d3 in the dcLNs. Data were analyzed by the two-way ANOVA with Tukey’s post hoc test or repeated two-way ANOVA with Tukey’s post hoc test. Data are mean ± SEM. n = 4 per group, *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 1 AQP4 deletion aggravates decreases of florescent tracer influx into, and efflux out of, the brain of 3-month-old APP/PS1 mice. a, b Schematic images for intracisternal injection and intrastriatal injection of TR-d3. c Representative images of serial coronal brain sections from 1.0 mm anterior to 1.8 mm posterior to bregma (upper panel) and high magnification micrographs of the sections at the level of 0.5 mm anterior to bregma (low panel) showing TR-d3 penetration into the brain parenchyma at 30 min after intracisternal injection. d High magnification micrographs of the cortex showing TR-d3 penetration into the perivascular space (star) and adjacent brain parenchyma (dotted line) at 30 min after intracisternal injection. e Representative images of serial coronal brain sections from 1.0 mm anterior to 1.8 mm posterior to bregma (upper panel) and high magnification micrographs of the sections at the level of 0.2 mm anterior to bregma (low panel) showing residue of TR-d3 within the striatum at 1 h after intracisternal injection. f Low (upper panel) and high (low panel) magnification micrographs of sections of the dcLNs showing TR-d3 distribution at 1 h after intracisternal injection. g Quantification of intracisternally injected TR-d3 area fraction in the brain. h Quantification of the mean fluorescence intensity of TR-d3 in the cerebral cortex after intracisternal injection. i Quantification of intrastriatally injected TR-d3 area in the brain. j Quantification of intrastriatal injected TR-d3 in the dcLNs. Data were analyzed by the two-way ANOVA with Tukey’s post hoc test or repeated two-way ANOVA with Tukey’s post hoc test. Data are mean ± SEM. n = 4 per group, *p < 0.05; **p < 0.01; ***p < 0.001CC BY · PubMed Central
Fig. 2 AQP4 deletion increased intraneuronal Aβ and IDE activity without causing plaque formation in the cerebral cortex of 3-month-old APP/PS1 mice. a, b Brain sections stained by thioflavine-S florescence and Aβ1–40 immunohistochemistry showing no Aβ plaque disposition. c Double immunofluorescence for total-Aβ and NeuN. There was increased Aβ immunostaining in cerebral neurons of AQP4−/−APP/PS1 mice compared to those in APP/PS1 controls. d, g Representative Western blot bands and densitometry analysis of APP, CTF-β, and Aβ monomer/oligomers. e, h Representative Western blot bands and densitometry analysis of BACE1, PS1, LRP1, IDE, and NEP in the cortex. f Quantification of Aβ-positive area fraction. i Enzyme activity assay of NEP and IDE. Data in f were analyzed by Student’s t test; other data were analyzed by the two-way ANOVA with Tukey’s post hoc test. Data are mean ± SEM. n = 4 per group. *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 2 AQP4 deletion increased intraneuronal Aβ and IDE activity without causing plaque formation in the cerebral cortex of 3-month-old APP/PS1 mice. a, b Brain sections stained by thioflavine-S florescence and Aβ1–40 immunohistochemistry showing no Aβ plaque disposition. c Double immunofluorescence for total-Aβ and NeuN. There was increased Aβ immunostaining in cerebral neurons of AQP4−/−APP/PS1 mice compared to those in APP/PS1 controls. d, g Representative Western blot bands and densitometry analysis of APP, CTF-β, and Aβ monomer/oligomers. e, h Representative Western blot bands and densitometry analysis of BACE1, PS1, LRP1, IDE, and NEP in the cortex. f Quantification of Aβ-positive area fraction. i Enzyme activity assay of NEP and IDE. Data in f were analyzed by Student’s t test; other data were analyzed by the two-way ANOVA with Tukey’s post hoc test. Data are mean ± SEM. n = 4 per group. *p < 0.05; **p < 0.01; ***p < 0.001CC BY · PubMed Central
Fig. 3 Increased microglial activation, and Aβ plaque deposition following eliminating microglia in AQP4−/−/APP/PS1 mice. a Double immunofluorescence for total-Aβ and Iba1. Microglia were apparently activated in the cerebral cortex of AQP4−/−/APP/PS1 mice, compared to those in APP/PS1 controls. Dot-like signals of total-Aβ (arrowheads) were frequently observed in AQP4−/−/APP/PS1 microglial cells. b, c Quantification of Iba1-positive, and iba1 and total-Aβ double-positive area fraction in the cerebral cortex, respectively. d, f Double immunofluorescence and quantification for CD68 and Iba1. CD68-positive microglia (arrowheads) were apparently increased in the cerebral cortex of AQP4−/−/APP/PS1 mice, compared to those in APP/PS1 controls. e, g Double immunofluorescence and quantification for Lamp1 and Iba1. Lamp1-positive microglia (arrowheads) were also apparently increased in the cerebral cortex of AQP4−/−/APP/PS1 mice. h, i Triple immunofluorescence and quantification for Lamp1, total-Aβ, and Iba1. Aβ immunoreactive products were restrictively localized to Lamp1-positive lysosome of Iba1-positive microglia. j Iba1 positive microglia were almost eliminated in both APP/PS1 mice and AQP4−/−/APP/PS1 with clodronate liposome treatment. k, l Brain sections stained by thioflavine-S and Aβ1–40, respectively. Aβ plaque disposition was present at the cerebral cortex of AQP4−/−/APP/PS1 mice received local injection of clodronate liposomes. m Double immunofluorescence for total-Aβ and NeuN. Total-Aβ immunoreactivity (arrowheads) was increased within the cytoplasm of NeuN positive neurons of AQP4−/−/APP/PS1 mice injected clodronate liposomes. n–s Quantification of Iba1, thioflavine-S, Aβ1–40, and total-Aβ-positive area fraction or number in the cerebral cortex, respectively. Data in c, f, g, and i were analyzed by Student’s t test and in b, n, q, and s were analyzed by the two-way ANOVA with Newman-Keuls post hoc test. Data are mean ± SEM, n = 4 per group, *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 3 Increased microglial activation, and Aβ plaque deposition following eliminating microglia in AQP4−/−/APP/PS1 mice. a Double immunofluorescence for total-Aβ and Iba1. Microglia were apparently activated in the cerebral cortex of AQP4−/−/APP/PS1 mice, compared to those in APP/PS1 controls. Dot-like signals of total-Aβ (arrowheads) were frequently observed in AQP4−/−/APP/PS1 microglial cells. b, c Quantification of Iba1-positive, and iba1 and total-Aβ double-positive area fraction in the cerebral cortex, respectively. d, f Double immunofluorescence and quantification for CD68 and Iba1. CD68-positive microglia (arrowheads) were apparently increased in the cerebral cortex of AQP4−/−/APP/PS1 mice, compared to those in APP/PS1 controls. e, g Double immunofluorescence and quantification for Lamp1 and Iba1. Lamp1-positive microglia (arrowheads) were also apparently increased in the cerebral cortex of AQP4−/−/APP/PS1 mice. h, i Triple immunofluorescence and quantification for Lamp1, total-Aβ, and Iba1. Aβ immunoreactive products were restrictively localized to Lamp1-positive lysosome of Iba1-positive microglia. j Iba1 positive microglia were almost eliminated in both APP/PS1 mice and AQP4−/−/APP/PS1 with clodronate liposome treatment. k, l Brain sections stained by thioflavine-S and Aβ1–40, respectively. Aβ plaque disposition was present at the cerebral cortex of AQP4−/−/APP/PS1 mice received local injection of clodronate liposomes. m Double immunofluorescence for total-Aβ and NeuN. Total-Aβ immunoreactivity (arrowheads) was increased within the cytoplasm of NeuN positive neurons of AQP4−/−/APP/PS1 mice injected clodronate liposomes. n–s Quantification of Iba1, thioflavine-S, Aβ1–40, and total-Aβ-positive area fraction or number in the cerebral cortex, respectively. Data in c, f, g, and i were analyzed by Student’s t test and in b, n, q, and s were analyzed by the two-way ANOVA with Newman-Keuls post hoc test. Data are mean ± SEM, n = 4 per group, *p < 0.05; **p < 0.01; ***p < 0.001CC BY · PubMed Central
Fig. 4 AQP4 deletion increased intraneuronal apoE aggregation in 3-month-old AQP4−/−/APP/PS1 mice. a, b Representative bands of Western blot and densitometry analysis of apoE. c–e Double immunofluorescence of apoE/GS, apoE/NeuN, and apoE/total-Aβ, respectively. Note that AQP4 deletion in APP/PS1 mice increased apoE immunoreactive intensity in GS-positive astrocytes and NeuN-positive neurons. ApoE and total-Aβ double immunoreactive products were also accumulated. f–i Quantification of apoE, apoE/GS, apoE/NeuN, and apoE/total-Aβ-positive area fraction in the cerebral cortex, respectively. Data in i were analyzed by Student’s t test; other data were analyzed by the two-way ANOVA with Newman-Keuls post hoc test. Data are mean ± SEM, n = 4 per group, *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 4 AQP4 deletion increased intraneuronal apoE aggregation in 3-month-old AQP4−/−/APP/PS1 mice. a, b Representative bands of Western blot and densitometry analysis of apoE. c–e Double immunofluorescence of apoE/GS, apoE/NeuN, and apoE/total-Aβ, respectively. Note that AQP4 deletion in APP/PS1 mice increased apoE immunoreactive intensity in GS-positive astrocytes and NeuN-positive neurons. ApoE and total-Aβ double immunoreactive products were also accumulated. f–i Quantification of apoE, apoE/GS, apoE/NeuN, and apoE/total-Aβ-positive area fraction in the cerebral cortex, respectively. Data in i were analyzed by Student’s t test; other data were analyzed by the two-way ANOVA with Newman-Keuls post hoc test. Data are mean ± SEM, n = 4 per group, *p < 0.05; **p < 0.01; ***p < 0.001CC BY · PubMed Central
Fig. 5 AQP4 deletion increased intraneuronal apoE aggregation in 3-month-old AQP4−/−/APP/PS1 mice. a, b Representative bands of Western blot and densitometry analysis of apoE and LRP1. c Immunofluorescence of apoE. ApoE expression was significantly decreased in the cerebral cortex following local injection of AAV encoding apoE siRNAs. d Quantification of apoE-positive area fraction in the cerebral cortex. e Double immunofluorescence of NeuN and total-Aβ. Total-Aβ expression was also obviously decreased in the cerebral cortex following local injection of AAV encoding apoE siRNAs. f, g Quantification of total-Aβ and NeuN/total-Aβ-positive area fraction in the cerebral cortex, respectively. h, i Representative bands of Western blot and densitometry analysis of APP, CTF-β, and Aβ monomers/oligomers in the cortex after local injection of AAV encoding apoE siRNAs. Data were analyzed by two-way ANOVA with Newman-Keuls post hoc test. Data are mean ± SEM, n = 4 per group, *p < 0.05; ***p < 0.001
Fig. 5 AQP4 deletion increased intraneuronal apoE aggregation in 3-month-old AQP4−/−/APP/PS1 mice. a, b Representative bands of Western blot and densitometry analysis of apoE and LRP1. c Immunofluorescence of apoE. ApoE expression was significantly decreased in the cerebral cortex following local injection of AAV encoding apoE siRNAs. d Quantification of apoE-positive area fraction in the cerebral cortex. e Double immunofluorescence of NeuN and total-Aβ. Total-Aβ expression was also obviously decreased in the cerebral cortex following local injection of AAV encoding apoE siRNAs. f, g Quantification of total-Aβ and NeuN/total-Aβ-positive area fraction in the cerebral cortex, respectively. h, i Representative bands of Western blot and densitometry analysis of APP, CTF-β, and Aβ monomers/oligomers in the cortex after local injection of AAV encoding apoE siRNAs. Data were analyzed by two-way ANOVA with Newman-Keuls post hoc test. Data are mean ± SEM, n = 4 per group, *p < 0.05; ***p < 0.001CC BY · PubMed Central
Fig. 6 Schematic diagram of our working model for the glymphatic system mediated Aβ and apoE clearance and microglia mediated Aβ degradation synergistically preventing Aβ plague formation in the early stages of the AD mouse model. 3-month-old APP/PS1 mice show extracellular and intraneuronal accumulation of Aβ because of the long-term of expression transgenic APP/PS1. Excessive interstitial Aβ may result in activation of astrocytes with loss of perivascular AQP4 polarization, subsequently impairing glymphatic clearance of Aβ and apoE. On the other hand, microglia undergo activation and promote Aβ clearance, potentially preventing Aβ plaque formation. AQP4 deletion exacerbates glymphatic transport impairment and intraneuronal accumulation of Aβ and apoE, but Aβ plaques have not yet aggregated, which may be related to the increase of phagocytosis of reactive microglia
Fig. 6 Schematic diagram of our working model for the glymphatic system mediated Aβ and apoE clearance and microglia mediated Aβ degradation synergistically preventing Aβ plague formation in the early stages of the AD mouse model. 3-month-old APP/PS1 mice show extracellular and intraneuronal accumulation of Aβ because of the long-term of expression transgenic APP/PS1. Excessive interstitial Aβ may result in activation of astrocytes with loss of perivascular AQP4 polarization, subsequently impairing glymphatic clearance of Aβ and apoE. On the other hand, microglia undergo activation and promote Aβ clearance, potentially preventing Aβ plaque formation. AQP4 deletion exacerbates glymphatic transport impairment and intraneuronal accumulation of Aβ and apoE, but Aβ plaques have not yet aggregated, which may be related to the increase of phagocytosis of reactive microgliaCC BY · PubMed Central

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Metadata

Authors
Feng, W., Zhang, Y., Wang, Z., Xu, H., Wu, T., Marshall, C., Gao, J., & Xiao, M.
Affiliations
  • Nanjing Brain Hospital (CN)
  • Jiangsu Province Hospital (CN)
  • University of Kentucky (US)
Corresponding author
  • Gao, Junying
  • Xiao, Ming
ORCID
Venue
Alzheimer's Research & Therapy
Publisher
Springer Science and Business Media LLC
Published
2020-10-02
Volume
12
Issue
1
Pages
125-125
ISSN
1758-9193 (ISSN-L)
DOI
10.1186/s13195-020-00688-1
PMC
PMC7532614
OpenAlex
W3090445757
Cited by
150
References
65
Citations by year
2026: 20 · 2025: 27 · 2024: 27 · 2023: 35 · 2022: 25 · 2021: 15 · 2020: 1
FWCI
11.39
Type
journal article
Language
en
Open access
Licence
Locations
  • Alzheimer s Research & Therapy (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • UKnowledge (University of Kentucky) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Natural Science Foundation of China (81671070, 81772454)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Clinical Neurology › Cerebrospinal fluid and hydrocephalus
Keywords
Alzheimer's disease, amyloid-beta, glymphatic system, microglia, aquaporin-4, apolipoprotein E, amyloid plaque formation, APP/PS1 mice, clodronate liposomes, microglial phagocytosis, amyloid clearance, astrocytes
MeSH terms
Alzheimer Disease, Animals, Disease Models, Animal, Mice, Transgenic, Amyloid beta-Peptides, Amyloid beta-Protein Precursor, Microglia, Mice, Presenilin-1, Plaque, Amyloid

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