Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 11, 42
eCollection

Emergence of Microglia Bearing Senescence Markers During Paralysis Progression in a Rat Model of Inherited ALS

Affiliations

Emergence of Microglia Bearing Senescence Markers During Paralysis Progression in a Rat Model of Inherited ALS

Emiliano Trias et al. Front Aging Neurosci.

Abstract

Age is a recognized risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive loss of motor neurons and neuroinflammation. A hallmark of aging is the accumulation of senescent cells. Yet, the pathogenic role of cellular senescence in ALS remains poorly understood. In rats bearing the ALS-linked SOD1G93A mutation, microgliosis contribute to motor neuron death, and its pharmacologic downregulation results in increased survival. Here, we have explored whether gliosis and motor neuron loss were associated with cellular senescence in the spinal cord during paralysis progression. In the lumbar spinal cord of symptomatic SOD1G93A rats, numerous cells displayed nuclear p16INK4a as well as loss of nuclear Lamin B1 expression, two recognized senescence-associated markers. The number of p16INK4a-positive nuclei increased by four-fold while Lamin B1-negative nuclei increased by 1,2-fold, respect to non-transgenic or asymptomatic transgenic rats. p16INK4a-positive nuclei and Lamin B1-negative nuclei were typically localized in a subset of hypertrophic Iba1-positive microglia, occasionally exhibiting nuclear giant multinucleated cell aggregates and abnormal nuclear morphology. Next, we analyzed senescence markers in cell cultures of microglia obtained from the spinal cord of symptomatic SOD1G93A rats. Although microglia actively proliferated in cultures, a subset of them developed senescence markers after few days in vitro and subsequent passages. Senescent SOD1G93A microglia in culture conditions were characterized by large and flat morphology, senescence-associated beta-Galactosidase (SA-β-Gal) activity as well as positive labeling for p16INK4a, p53, matrix metalloproteinase-1 (MMP-1) and nitrotyrosine, suggesting a senescent-associated secretory phenotype (SASP). Remarkably, in the degenerating lumbar spinal cord other cell types, including ChAT-positive motor neurons and GFAP-expressing astrocytes, also displayed nuclear p16INK4a staining. These results suggest that cellular senescence is closely associated with inflammation and motor neuron loss occurring after paralysis onset in SOD1G93A rats. The emergence of senescent cells could mediate key pathogenic mechanisms in ALS.

Keywords: ALS; SASP; aging; astrocytes; microglia; motor neurons; senescence.

Figures

FIGURE 1
FIGURE 1
Progressive change in senescence markers p16INK4a and Lamin B1 during paralysis progression in SOD1G93A rats ventral spinal cord. Representative confocal images showing the expression of p16INK4a (green) and Lamin B1 (red) by immunohistochemistry in the degenerating spinal cord of SOD1G93A animals and non-transgenic controls. (A) Progressive increase of the senescence marker p16INK4a staining (green) in nuclei from the ventral horn of the spinal cord in symptomatic rat during paralysis progression (white dotted lines indicate the separation of white and gray matter). The graph to the right shows the quantitative analysis of the p16INK4a-positive nuclei in the ventral spinal cord. Data are expressed as mean ± SEM; data were analyzed by Kruskal–Wallis followed by Dunn’s multiple comparison tests, p < 0.05 was considered statistically significant. Scale bar: 50 μm. (B) Confocal microphotographs showing the staining for nuclear Lamin B1 (red) as a marker of non-senescent cells among analyzed groups. The graph to the right shows the quantitative analysis of Lamin B1-positive nuclei in the ventral horn of the spinal cord. Note the progressive loss of nuclear Lamin B1 expression with disease progression. Data are expressed as mean ± SEM; data were analyzed by Kruskal–Wallis followed by Dunn’s multiple comparison tests, p < 0.05 was considered statistically significant. Scale bar: 50 μm. (C) The confocal images show co-expression of p16INK4a (green) p53 (red) nuclear staining (white arrows) in the ventral horn of degenerating spinal cord at 15d post-paralysis. The inset shows the nuclear localization of p53 in a subset of cells. Scale bars: 10 μm. (D) High magnification confocal image showing the loss of nuclear Lamin B1 (red) expression (white arrows) and Lamin B1 invaginations (asterisks) associated to nuclear misshape. Scale bar: 10 μm.
FIGURE 2
FIGURE 2
Nuclear p16INK4a and Lamin B1 expression in microglia during paralysis progression. (A) Confocal representative images showing the expression of the microglia marker Iba1 (red) and the senescence marker p16INK4a (green) in the non-transgenic, asymptomatic, onset and 15d paralysis SOD1G93A ventral spinal cord. The upper panels (low magnification) show the significant parallel increase of nuclear p16INK4a in Iba1-positive cells during the symptomatic stage of the disease as compared with non-transgenic animals or SOD1G93A asymptomatic stage. Lower panels show at high magnification images of p16INK4a-positive swollen microglia (white arrows) surrounding motor neurons (MTN). The graph below shows the quantitative analysis of the expression of p16INK4a- in Iba1-positive cells. Note the sharp increase of p16INK4a–positive microglia at 15d post-paralysis. Data are expressed as mean ± SEM; data were analyzed by Kruskal–Wallis followed by Dunn’s multiple comparison tests, p < 0.05 was considered statistically significant. Scale bars: 50 μm in low magnification panels and 10 μm in high magnification panels. (B) The confocal microphotograph shows a multinucleated microglia cluster expressing Iba1 (red) in a 15d paralysis rat. These Iba1-positive clusters express nuclear p16INK4a. Scale bar: 20 μm. (C) Representative confocal microphotograph of the ventral spinal cord showing nuclear Lamin B1 expression in Iba1-positive cells (arrowheads). Note the loss of Lamin B1 expression in a subpopulation of cells (arrows) at 15d post-paralysis. Scale bar: 20 μm. (D) The confocal microphotograph shows a cluster of multinucleated microglia where Lamin B1 expression is absent in several nuclei (DAPI) at 15d post-paralysis. Scale bar: 20 μm.
FIGURE 3
FIGURE 3
Nuclear p16INK4a expression in a subpopulation of spinal motor neurons and astrocytes. (A) Representative confocal microphotograph of the ventral spinal cord of SOD1G93A rats showing ChAT-positive (red) motor neurons at low (upper row) and high (lower row) magnifications. During the symptomatic phase of the disease, a subpopulation of neurons expresses nuclear p16INK4a (white arrows). Dotted white line separate white from gray matter. Scale bars: 50 μm for low magnification panels and 10 μm for high magnification panel. (B) Photomicrographs showing p16INK4a/GFAP stained lumbar spinal cord sections among groups. Low magnification panels (upper panels) show the notorious increase in the number of p16INK4a-/GFAP-positive cells in the symptomatic rats, as compared to low markers co-expression in asymptomatic or non-transgenic rats. Note the expression of p16INK4a marker in a subpopulation of astrocytes that surround motor neurons. Scale bars: 50 μm for low magnification panels and 10 μm for high magnification panel.
FIGURE 4
FIGURE 4
Senescence-associated β-Galactosidase activity in primary cultures of microglia from symptomatic SOD1G93A rats. (A) The scheme shows the procedure for adult microglia cell cultures from symptomatic SOD1G93A rats. The spinal cord was plated on p35 culture dishes and SA-b-Gal was measured at different time points. Senescent markers increase their expression after several days in culture. After 2 weeks in vitro, microglia transitioned to aberrant glial cells. These transformed cells were also analyzed for SA-β-Gal and senescence markers at different time points in culture. (B) The phase contrast microphotographs show SA-β-Gal staining after 2 days in vitro (DIV) and 12 DIV. The graph to the right shows the quantitative analysis of SA-β-Gal activity in cultured adult microglia at different time points. Data are expressed as mean ± SEM; data were analyzed by Kruskal–Wallis followed by Dunn’s multiple comparison tests, p < 0.05 was considered statistically significant. (C) SA-β-Gal activity analyzed by flow cytometry analysis. In the scatter diagram for the smaller population (inside white outline), R1 indicates the percentage of total population encompassed by this subset (45%). The gate for the smaller cell population indicates almost 8% of these cells are senescent. (D) The diagram shows the cell cycle analysis for the smaller cell population. (E) Scatter diagram for larger cell population (inside the white outline, R2). In the larger cell population, over 90% of the cells demonstrate SA-β-Gal activity. (F) The scatter diagram to the right shows the cell cycle analysis for the larger cell population.
FIGURE 5
FIGURE 5
Cultured adult microglia from SOD1G93A symptomatic rats express senescence markers. Immunocytochemistry analysis of senescence markers on microglia isolated from SOD1G93A symptomatic rats at 2 and 12DIV. (A) Isolated Iba1-positive microglia express nuclear p16INK4a, which expression increase after several days in culture as shown in the graph to the right. Data are expressed as mean ± SEM: data were analyzed by Mann–Whitney test, 2-tailed, p < 0.05 was considered statistically significant. Scale bar: 50 μm. (B) CD68-positive microglia express increasing levels of nuclear p53 in culture. The graph to the right shows the comparative quantitative analysis of p53 expression. Data are expressed as mean ± SEM: data were analyzed by Mann-Whitney test, 2-tailed, p < 0.05 was considered statistically significant. Scale bar: 50 μm. (C) After 12 DIV, SOD1G93A isolated microglia form Iba1-/CD68-positive multinucleated giant cells, which express several senescence markers such as p16INK4a, p53, and MMP1. Also, these multinucleated cells express high levels of NO2Tyr. Scale bars: 20 μm.
FIGURE 6
FIGURE 6
Potential mechanisms underlying the emergence of senescent phenotypes in ALS and pathophysiological consequences. Risk factors such as aging, mitochondrial damage, nitro-oxidative stress, and inflammation may induce the appearance of senescent glial cells in the surroundings of motor neurons bearing SASP. In turn, these cells may exacerbate inflammation and induce motor neuron toxicity through the secretion of soluble toxic factors. This scenario might lead to a pathogenic autotoxic loop promoting the spread of motor neuron pathology and disease progression.

Similar articles

See all similar articles

Cited by 2 PubMed Central articles

References

    1. Al-Chalabi A., Calvo A., Chio A., Colville S., Ellis C. M., Hardiman O., et al. (2014). Analysis of amyotrophic lateral sclerosis as a multistep process: a population-based modelling study. Lancet Neurol. 13 1108–1113. 10.1016/S1474-4422(14)70219-4 - DOI - PMC - PubMed
    1. Aloi M. S., Su W., Garden G. A. (2015). The p53 Transcriptional Network Influences Microglia Behavior and Neuroinflammation. Crit. Rev. Immunol. 35 401–415. - PMC - PubMed
    1. Appel S. H., Zhao W., Beers D. R., Henkel J. S. (2011). The microglial-motoneuron dialogue in ALS. Acta Myol. 30 4–8. - PMC - PubMed
    1. Arendt T., Rodel L., Gartner U., Holzer M. (1996). Expression of the cyclin-dependent kinase inhibitor p16 in Alzheimer’s disease. Neuroreport 7 3047–3049. - PubMed
    1. Baker D. J., Wijshake T., Tchkonia T., LeBrasseur N. K., Childs B. G., van de Sluis B., et al. (2011). Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479 232–236. 10.1038/nature10600 - DOI - PMC - PubMed
Feedback