p38MAPK pathway is activated and modulates IL-6 secretion in astrocytes.

<p>(<b>A</b>) 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>B</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), *<i>P</i><0.01 vs. senescent, Student’s t test.</p

Expression level of p16^INK4a is not elevated in adult cerebellar astrocytes.

<p>(<b>A</b>) Representative images of cerebellum from an AD patient showing intensely p16^INK4a positive granular layer. The Bergmann glia (arrowhead) and Purkinje neurons (arrow) are negative. Blue: DAPI; Green: GFAP; Red: p16^INK4a. (<b>B</b>) Bar diagram shows increased mean percentage of p16^INK4a-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 p16^INK4a-positive astrocytes in cerebellum is comparable to fetal cortical levels. *<i>P</i> = 0.04, Student’s t test, cerebellum vs. control cortices; **<i>P</i> = 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.</p

Senescent astrocytes secrete pro-inflammatory mediators that may constitute a SASP.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045069#s4" target="_blank">Materials and Methods</a>.</p

Mouse embryo fibroblasts from IGF-I-depleted mice show increased mitochondrial mass and DNA content.

<p>Mouse embryo fibroblasts (MEFs) were growth from IGF-I deficient mice or control animals as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012592#s4" target="_blank">materials and methods</a>. The relative mitochondria content in wild type and IGF-I-depleted mice measured by staining with the mitochondrial specific mitotracker green fluorescent dye as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012592#s4" target="_blank">Materials and Methods</a>. <b>A.</b> Mean mitochondrial mass of the cell populations at passage 2 and 10 is presented as analyzed by flow cytometery. Differences between the mitochondrial mass in the IGF-I deficient mice and controls was significant (<i>P</i><0.01 at passage 2 and <i>P</i><0.05 at passage 10). <b>B.</b> Relative mitochondrial DNA content at passage 2 is presented. The experiment presented is representative of the results of 2 independent measurements on 4 DNA isolates using independent primer sets <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012592#pone.0012592-Guo1" target="_blank">[45]</a> that amplify mitochondrial and nuclear DNA. The difference in mitochondrial DNA content was significant (P<0.05).</p

Astrocytes trigger senescence in response to Aβ_1–42.

<p>(<b>A</b>) Representative images of astrocytes treated with conditioned media from 7PA2 Aβ-secreting Chinese Hamster Ovary (CHO) cells or CHO control cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045069#pone.0045069.s003" target="_blank">Figure S2</a>. Graph shows relative amount of SA β-gal-positive cells from 3 independent experiments, *<i>P</i> = 0.039. (<b>B</b>) Representative images of astrocytes treated with oligomerized synthetic Aβ_1–42 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, *<i>P</i> = 0.017, Aβ_1–42 (5 µM and **<i>P</i> = 0.009, Aβ_1–42 (10 µM) vs. control. (<b>C</b>) Immunoblot for p16^INK4a expression in astrocytes treated with 5 µM Aβ_1–42 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 p16^INK4a:β-actin, *<i>P</i> = 0.02. For all experiments, error bars represent SD and Student’s <i>t</i>-test was used to determine significance.</p

Increased frequency of senescent astrocytes during brain aging and AD.

<p>p16^INK4a-positive astrocytes were identified in formalin-fixed, paraffin-embedded frontal cortex sections by double immunofluorescence with p16^INK4a/GFAP. (<b>A</b>) Double immunofluorescence with p16^INK4a/GFAP showing increased p16^INK4a-positive astrocytes with increased age and AD (representative images). Blue: DAPI; green: GFAP; red: p16^INK4a. Arrows indicate p16^INK4a-positive astrocytes. (<b>B</b>) Bar diagram shows increased mean p16^INK4a-positive astrocytes in frontal cortices from non-AD adult subjects (35–50 years, <i>n</i> = 6; 78–90 years, <i>n</i> = 11) as compared to fetal autopsy tissue (<i>n</i> = 4). *<i>P</i> = 0.02, fetal vs. 35–50 year olds; **<i>P</i><10⁻⁶, fetal vs. 78–90 year olds; Student’s <i>t</i>-test. (<b>C</b>) Bar diagram shows increased mean p16^INK4a-positive astrocytes in frontal cortices from AD adult subjects (<i>n</i> = 15) compared to non-AD adult control subjects (<i>n</i> = 25) of similar ages; and fetal controls (<i>n</i> = 4). ^*<i>P</i> = 0.02, AD vs. adult controls; ***<i>P</i><10⁻⁷, AD vs. fetal controls, and **<i>P</i><10⁻⁶, adult controls vs. fetal controls. Error bars represent SD and Student’s <i>t</i>-test was used to determine significance. At least 200 cells were counted per slide.</p

IGF-I treatment increases mitochondrial depolarization.

<p>WI-38 fibroblasts were maintained for 14 days in MCDB 105 medium, MCDB 105 medium with IGF-I (40 ng/ml), or MCDB 105 medium with EGF (40 ng/ml). Medium with or without growth factors was replenished every 3 days and cells were stained for mitochondrial potential at that time as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012592#s4" target="_blank">materials and methods</a>. Cells with depolarized mitochondria were visualized by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012592#s4" target="_blank">Material and Methods</a>. <b>A.</b> Percentage of cells with depolarized mitochondria as assessed by JC-1 staining (*, <i>P</i><0.001). <b>B.</b> Representative dot blot of JC-1-stained cultures in MCDB 105 with or without IGF-I or EGF. Y-axis, fluorescence at 590 nm; X-axis, fluorescence at 525 nm. A downward shift on the X-axis is indicative of mitochondrial membrane depolarization.</p

MMP-1 expression parallels p16^INK4a expression in human astrocytes in vivo.

<p>(<b>A</b>) 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>B</b>) Bar diagram shows positive correlation between MMP-1 and p16^INK4a expression in human frontal cortex, independent of diagnosis, <i>P</i> = 0.02, Spearman’s correlation coefficient = 0.574.</p

IGF-I decreases long-term viability of human fibroblasts.

<p>Long-term IGF-I treatment reduces colony formation potential. Colony forming assays were performed on cells that had been maintained in either MCDB 105 medium without additions or with IGF-I (40 ng/ml) for 2 weeks. Cells were seeded in full growth medium to allow colony growth and results are presented in panel <b>A</b>. Bars are number of colonies per 3×10³ cells plated (*, <i>P</i><0.01) <b>B.</b> Representative micrograph (20X) of senescence-associated β-galactosidase staining of fibroblast colonies. <b>C.</b> Crystal violet stained colonies of plates seeded with 3×10³ cells for colony forming assays.</p

Impairment of autophagy increases mitochondrial depolarization.

<p>WI-38 fibroblasts and WI-38 cells expressing either an shRNA construct targeting Atg5 (shAtg5) or the same targeting vector expressing a scrambled sequence (Scramble) were maintained for 14 days in MCDB 105 medium. As a control, parallel cultures were maintained in MCBD 105, or MCDB 105 medium with IGF-I (40 ng/ml). Medium with or without growth factor was replenished every 3 days. Cells were stained for mitochondrial potential at that time as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012592#s4" target="_blank">materials and methods</a>. Cells with depolarized mitochondria were visualized by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012592#s4" target="_blank">Material and Methods</a>. Differences in the percent of cells with depolarized mitochondria between cell populations were significant for shAtg5 versus scramble or control cells (*, <i>P</i><0.05). The difference in the percent of cells with depolarized mitochondria between IGF-I treated and either control or scramble was also significant (*, <i>P</i><0.05).</p