Figure 1.
Astrocytes trigger senescence in response to Aβ1–42.
(A) Representative images of astrocytes treated with conditioned media from 7PA2 Aβ-secreting Chinese Hamster Ovary (CHO) cells or CHO control cells as described in Figure S2. Graph shows relative amount of SA β-gal-positive cells from 3 independent experiments, *P = 0.039. (B) Representative images of astrocytes treated with oligomerized synthetic Aβ1–42 for 2 hours and assayed for SA β-gal activity after 3 days. Graph shows relative levels of SAβ-gal–positive cells from 3 independent experiments, *P = 0.017, Aβ1–42 (5 µM and **P = 0.009, Aβ1–42 (10 µM) vs. control. (C) Immunoblot for p16INK4a expression in astrocytes treated with 5 µM Aβ1–42 for 24 hours. Lysate was collected 4 days after treatment initiation. β-actin serves as a loading control. Graph depicts normalized optical density of the ratio of p16INK4a:β-actin, *P = 0.02. For all experiments, error bars represent SD and Student’s t-test was used to determine significance.
Figure 2.
Senescent astrocytes secrete pro-inflammatory mediators that may constitute a SASP.
Pre-senescent and senescent astrocytes were incubated in serum-free MCDB105 media. Conditioned media was collected after 48 hours and analyzed by antibody array (RayBiotech, Inc.; Norcross, GA) for pro-inflammatory factors as described in Materials and Methods.
Figure 3.
Increased frequency of senescent astrocytes during brain aging and AD.
p16INK4a-positive astrocytes were identified in formalin-fixed, paraffin-embedded frontal cortex sections by double immunofluorescence with p16INK4a/GFAP. (A) Double immunofluorescence with p16INK4a/GFAP showing increased p16INK4a-positive astrocytes with increased age and AD (representative images). Blue: DAPI; green: GFAP; red: p16INK4a. Arrows indicate p16INK4a-positive astrocytes. (B) Bar diagram shows increased mean p16 INK4a-positive astrocytes in frontal cortices from non-AD adult subjects (35–50 years, n = 6; 78–90 years, n = 11) as compared to fetal autopsy tissue (n = 4). *P = 0.02, fetal vs. 35–50 year olds; **P<10−6, fetal vs. 78–90 year olds; Student’s t-test. (C) Bar diagram shows increased mean p16 INK4a-positive astrocytes in frontal cortices from AD adult subjects (n = 15) compared to non-AD adult control subjects (n = 25) of similar ages; and fetal controls (n = 4). *P = 0.02, AD vs. adult controls; ***P<10−7, AD vs. fetal controls, and **P<10−6, adult controls vs. fetal controls. Error bars represent SD and Student’s t-test was used to determine significance. At least 200 cells were counted per slide.
Figure 4.
Expression level of p16INK4a is not elevated in adult cerebellar astrocytes.
(A) Representative images of cerebellum from an AD patient showing intensely p16INK4a positive granular layer. The Bergmann glia (arrowhead) and Purkinje neurons (arrow) are negative. Blue: DAPI; Green: GFAP; Red: p16INK4a. (B) Bar diagram shows increased mean percentage of p16INK4a-positive astrocytes in formalin-fixed, paraffin-embedded cortical sections from AD subjects (n = 3) and control subjects (n = 3) compared to cerebellum from both AD and aged-matched controls (n = 6). The mean percentage of p16INK4a-positive astrocytes in cerebellum is comparable to fetal cortical levels. *P = 0.04, Student’s t test, cerebellum vs. control cortices; **P = 0.004, Student’s t test, cerebellum vs. AD cortices. Error bars represent SD At least 200 cells per slide were counted for the cortical sections, and at least 100 cells per slide were counted for the cerebellar sections.
Figure 5.
MMP-1 expression parallels p16INK4a expression in human astrocytes in vivo.
(A) Double immunofluorescence with MMP-1/GFAP in AD and a similar-aged control (representative images). Blue: DAPI; red: GFAP; green: MMP-1. Arrows indicate MMP-1–positive astrocytes. Inset shows magnified image of MMP-1-positive astrocytes (yellow: cytoplasmic). (B) Bar diagram shows positive correlation between MMP-1 and p16INK4a expression in human frontal cortex, independent of diagnosis, P = 0.02, Spearman’s correlation coefficient = 0.574.
Figure 6.
p38MAPK pathway is activated and modulates IL-6 secretion in astrocytes.
(A) Western blots depict the levels of total and phosphorylated p38MAPK and heat shock protein 27 (Hsp27) in pre-senescent and H2O2-induced senescent astrocytes with β-actin as loading control. (B) Pre-senescent and senescent astrocytes were treated with 10 µM SB-203580 or DMSO as a control for 48 hours prior to incubation in serum-free MCDB105 media. Conditioned media was collected after 24 hours and IL-6 was analyzed by ELISA (R&D Systems, Inc.; Minneapolis, MN) and normalized to cell number. Graph depicts the relative level of IL-6 (n = 3), *P<0.01 vs. senescent, Student’s t test.