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. 2021 Jun 15;18(1):139.
doi: 10.1186/s12974-021-02191-2.

Microglia RAGE exacerbates the progression of neurodegeneration within the SOD1G93A murine model of amyotrophic lateral sclerosis in a sex-dependent manner

Michael MacLean  1 , Judyta Juranek  1   2 , Swetha Cuddapah  1 , Raquel López-Díez  1 , Henry H Ruiz  1 , Jiyuan Hu  3 , Laura Frye  1 , Huilin Li  3 , Paul F Gugger  1 , Ann Marie Schmidt  4
Affiliations

Microglia RAGE exacerbates the progression of neurodegeneration within the SOD1G93A murine model of amyotrophic lateral sclerosis in a sex-dependent manner

Michael MacLean et al. J Neuroinflammation. .

Abstract

Background: Burgeoning evidence highlights seminal roles for microglia in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). The receptor for advanced glycation end products (RAGE) binds ligands relevant to ALS that accumulate in the diseased spinal cord and RAGE has been previously implicated in the progression of ALS pathology.

Methods: We generated a novel mouse model to temporally delete Ager from microglia in the murine SOD1G93A model of ALS. Microglia Ager deficient SOD1G93A mice and controls were examined for changes in survival, motor function, gliosis, motor neuron numbers, and transcriptomic analyses of lumbar spinal cord. Furthermore, we examined bulk-RNA-sequencing transcriptomic analyses of human ALS cervical spinal cord.

Results: Transcriptomic analysis of human cervical spinal cord reveals a range of AGER expression in ALS patients, which was negatively correlated with age at disease onset and death or tracheostomy. The degree of AGER expression related to differential expression of pathways involved in extracellular matrix, lipid metabolism, and intercellular communication. Microglia display increased RAGE immunoreactivity in the spinal cords of high AGER expressing patients and in the SOD1G93A murine model of ALS vs. respective controls. We demonstrate that microglia Ager deletion at the age of symptomatic onset, day 90, in SOD1G93A mice extends survival in male but not female mice. Critically, many of the pathways identified in human ALS patients that accompanied increased AGER expression were significantly ameliorated by microglia Ager deletion in male SOD1G93A mice.

Conclusions: Our results indicate that microglia RAGE disrupts communications with cell types including astrocytes and neurons, intercellular communication pathways that divert microglia from a homeostatic to an inflammatory and tissue-injurious program. In totality, microglia RAGE contributes to the progression of SOD1G93A murine pathology in male mice and may be relevant in human disease.

Keywords: ALS; Astrocytes; Macrophage; Microglia; Neurodegeneration; RAGE.

PubMed Disclaimer

Conflict of interest statement

A.M.S is an inventor on awarded (US 9,353,3078, US 9364472) and pending (US 16/094270, Europe 177864360.0) patent applications assigned to NYU Grossman Medical Center.

Figures

Fig. 1
Fig. 1
Analysis of human ALS patient RNA-seq data implicates the RAGE pathway in modulating disease. a Normalized AGER counts per million mapped reads across patient groups separated by sex (no significant sex-dependent differences). b Barcode plot illustrating gene set enrichment of the “AGE-RAGE signaling pathway in diabetic complications” between ALS patients and non-neurological controls. The snake illustrates the enrichment and upregulation of the gene set in ALS patients. c Correlation of AGER expression (x-axis) and age at onset in years (y-axis). Regression line shown. Coefficient = − 2.449, p = 0.07, adjusted r2 = 0.032. d Correlation of AGER expression (x-axis) and age at death/tracheostomy in years (y-axis). Regression line shown. Coefficient = − 2.609, p = 0.037, adjusted r2 = 0.045. e Histogram of normalized AGER expression in ALS patients. Red boxes notate the highest 10% and the lowest 10% of AGER-expressing patients. f Representative image of regions of interest for RAGE staining in the ventral horn of a high and low AGER patient cervical spinal cord. Scale bar: 25 μm. g Quantification of RAGE+ area. In a: from left to right N = 38, 5, 38, 6. In b: N = 76 ALS patients and N = 11 non-neurological control patients. In c–e: N = 76 ALS patients. In f, g: N = 4 high AGER and 3 low AGER patients. Independent two sample two-sided t test. *p = 0.0349
Fig. 2
Fig. 2
AGER expression changes coincide with alterations in overlap of microglia and RAGE protein. a Representative images of regions of interest for RAGE, IBA1, GFAP, and NeuN staining in the ventral horn of high and low AGER patient cervical spinal cord and anterior white matter. Scale bar: 25 μm. b Quantification of IBA1+ area. c Quantification of GFAP+ area. d Quantification of overlap of IBA1+ RAGE+ area. e Quantification of overlap of GFAP+ RAGE+ area. f Quantification of overlap of NeuN+ RAGE+ area. N = 4 high AGER patients and N = 3 low AGER patients. Mean ± SD. In b–e: two-way ANOVA with post-hoc Tukey’s test. In f: Mann-Whitney U test. *p = 0.0102, ***p = 0.0009
Fig. 3
Fig. 3
SOD1G93A mice exhibit increased RAGE+ microglia at the age of 120 days. a Representative images of CD11B and RAGE staining in the ventral horn of SOD1G93A mouse lumbar spinal cord. Scale bar: 50 μm. b Quantification of overlap of CD11B+ RAGE+ area. N = 3 male WT mice, N = 4 male SOD1G93A mice, N = 4 female WT mice, and N = 4 female SOD1G93A mice. Mean ± SD. Two-way ANOVA with post-hoc Tukey’s test. *p = 0.0192, ****p < 0.0001
Fig. 4
Fig. 4
Study design and validation of microglia Ager deletion. a Timing of Tamoxifen (TAM) administration and behavioral assessments of animals. b Quantification of Ager transcripts in primary CD11B+ isolates by RT-qPCR thirty days after TAM administration. c Representative images of IBA1 and RAGE staining in the ventral horn of SOD1G93A mouse lumbar spinal cord at end-stage. Scale bar: 50 μm. d Quantification of IBA1 and RAGE overlap area. Mean ± SD. In b, N = 3 Agerfl/fl Cx3cr1+/+ mice and N = 4 Agerfl/fl Cx3cr1Cre/+ mice. In c, d, N = 6 SOD1G93A Agerfl/fl Cx3cr1Cre/+ mice, N = 8 SOD1G93A Ager+/+ Cx3cr1Cre/+ mice. Independent two sample two-sided t test. **p = 0.0031, *p = 0.0437
Fig. 5
Fig. 5
Microglia Ager deletion exerts sex-dependent effects on survival in SOD1G93A mice. a Kaplan–Meier estimates of percent of male mice surviving plotted vs. age (days). N = 16 SOD1G93A Agerfl/fl Cx3cr1Cre/+ mice, and N = 13 SOD1G93A Ager+/+ Cx3cr1Cre/+ mice. b Kaplan–Meier estimates of percent of female mice surviving plotted vs. age (days). N = 15 SOD1G93A Agerfl/fl Cx3cr1Cre/+ mice, and N = 14 SOD1G93A Ager+/+ Cx3cr1Cre/+ mice. The Logrank test was performed to compare the survival distributions between groups. *p = 0.0496
Fig. 6
Fig. 6
SOD1G93A mice with microglia Ager deletion exhibit sex-dependent effects on pathology. a Latency to fall as measured by hanging wire test in male mice. b Latency to fall as measured by hanging wire test in female mice. c Normalized body weight to weight at tamoxifen administration of male mice. d Normalized body weight to weight at tamoxifen administration of female mice. Dashed lines indicate age at tamoxifen administration. Mean ± SEM. e Representative images of NeuN and DAPI staining in the lumbar spinal cord ventral horn. Scale bar: 50 μm. f Quantification of NeuN+ DAPI+ cell number. Mean ± SD. In a, c (male mice): N = 16 SOD1G93A Agerfl/fl Cx3cr1Cre/+ mice, and N = 13 SOD1G93A Ager+/+ Cx3cr1Cre/+ mice. In b, d (female mice): N = 15 SOD1G93A Agerfl/fl Cx3cr1Cre/+ mice, and N = 14 SOD1G93A Ager+/+ Cx3cr1Cre/+ mice. In e, f (male mice): N = 4 SOD1G93A Agerfl/fl Cx3cr1Cre/+ mice, and N = 5 SOD1G93A Ager+/+ Cx3cr1Cre/+ mice. In a–d: independent two-sample two-sided t test was used to assess the group difference at each time point. Mixed effects analysis was utilized to evaluate effects of genotype, time, and the interaction between genotype and time. In a: genotype*time interaction effect: p = 0.0378. In c: genotype effect: p = 0.0157, genotype*time interaction effect: p = 0.0067. In d: genotype*time interaction effect p = 0.0134. In f: independent two sample two-sided t test. In f: *p = 0.0224
Fig. 7
Fig. 7
Differential expression analysis between male SOD1G93A mice with microglia devoid of Ager and Cre-expressing controls. Volcano plot displaying differentially expressed genes between SOD1G93A Agerfl/fl Cx3cr1Cre/+ and SOD1G93A Ager+/+ Cx3cr1Cre/+ mice. The y-axis is –log10(p value) and x-axis is the Log2 fold change of each gene analyzed. Red colored genes are significantly differentially expressed with an FDR < 0.05. The top twenty significant genes are labeled. N = 4 independent mice/group
Fig. 8
Fig. 8
Microglia Ager deletion in male SOD1G93A mice reduces the accumulation of disease-associated microglia at end-stage. a Representative images of CLEC7A staining in the lumbar spinal cord ventral horn of the indicated mouse groups. Scale bar: 50 μm. b Quantification of CLEC7A+ area. c Quantification of CLEC7A+ DAPI+ cell number. Mean ± SD. N = 7 SOD1G93A Agerfl/fl Cx3cr1Cre/+ mice and N = 9 SOD1G93A Ager+/+ Cx3cr1Cre/+ mice. Independent two sample two-sided t test. In b, **p < 0.0048. In c, *p = 0.0154
Fig. 9
Fig. 9
Reactive A1 astrocytes are reduced in male SOD1G93A mice by microglia Ager deletion. a Representative images of GFAP and AMIGO2 staining in the lumbar spinal cord ventral horn of the indicated mouse groups. b Quantification of GFAP+ area. c Quantification of GFAP+ and AMIGO2+ overlap area. Mean ± SD. N = 7 SOD1G93A Agerfl/fl Cx3cr1Cre/+ mice, and N = 9 SOD1G93A Ager+/+ Cx3cr1Cre/+ mice. Independent two sample two-sided t test. In b, *p = 0.0132. In c, **p = 0.0076
Fig. 10
Fig. 10
Microglia Ager deletion in male SOD1G93A mice reduces microgliosis at 120 days of age. a Representative images of CD11B staining in the lumbar spinal cord ventral horn of the indicated mouse groups. b Quantification of CD11B+ area. c. Quantification of CD11B+ DAPI+ cell number. d Representative images of CLEC7A staining in the lumbar spinal cord ventral horn of the indicated mouse groups. e Quantification of CLEC7A+ area. Mean ± SD. N = 4 SOD1G93A Agerfl/fl Cx3cr1Cre/+ mice, N = 6 SOD1G93A Ager+/+ Cx3cr1Cre/+ mice. In b, c, independent two sample two-sided t test. In e, Mann-Whitney U test. In b, * p = 0.0253. In c, *p = 0.0212. In e, *p = 0.0381
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