Article Figures & Data
Figures
- Figure 1.
Aging does not alter levels of AIS proteins, total tau, and phosphorylated tau proteins. A, B, Amount of 480-kDa (A) and 270-kDa (B) isoforms of AnkG do not change between young, middle, and old rats. Note that the other isoforms of AnkG not analyzed are isoforms that are not AIS-specific. C, AnkG Western blottings were normalized to Ponceau S (C) staining for loading control. D, E, The amount of βIV-spectrin (D) and neurofascin (E) also remain unchanged during aging in rats. F, The level of axonal neurofilaments, as indicated by the SMI-312 antibody. The SMI-312 antibody is an axonal marker that labels phospho-neurofilaments in axons. SMI-312 levels are not changed with age, confirming that total axon content is similar across age groups. G, H, The total level of tau detected with Tau7 (G) and polyclonal R1 (H) do not change with age in the rat HP. I, The levels of PHF1, a phospho-epitope of tau, are not changed with age. J, The total levels of microtubules, labeled with βIII-tubulin, remained unchanged with advancing age. K, GFAP, an astrocytic marker known to increase with age, is significantly increased in the HP of old age rats compared to young and middle aged rats; one-way ANOVA, Tukey’s post hoc, *p < 0.05. L, Western blot band intensities are normalized to GAPDH, which did not change with age. All data are displayed as mean ± SEM.
- Figure 2.
Structural analysis of the AIS with AnkG reveals regional changes in the HP during aging. A, The total length of AISs in the HP estimated with the spaceballs stereology probe is unchanged across age groups. B-D, The Neurolucida AutoNeuron analysis reveals that the length (B), volume (C), and diameter (D) of the AISs across young, middle, and old aged rats is not changed in CA1, CA3, or the dentate gyrus of the HP. The volume and diameter of the AISs in the CA2 region were significantly reduced in the old age compared with the young rats (one-way ANOVA, Tukey’s post hoc, *p < 0.05). All data are displayed as mean ± SEM. E-G, Representative images of the AISs (AnkG-positive immunostain, brown) in the dentate gyrus of the HP at ages 4 (E), 14 (G), and 24 (I) months, and the corresponding Neurolucida AutoNeuron tracings (F, H, and J; multicolor overlay to visually differentiate individual AISs). Scale bar, 20 µm (E-I).
- Figure 3.
Optical density measurement of tau immunofluorescence in the somata and axons of HP neurons shows no changes across age. A, The intensity of somatic tau (using Tau7, a total tau antibody) in neurons in the CA1, CA2, CA3, and dentate regions of the HP shows no change with advancing age. B, No change in the regional intensity of tau is detected in the axon-enriched strata of the HP (i.e., the fimbria, alveus, stratum lacunosum, and stratum moleculare). All data are displayed as mean ± SEM. C-E, Representative images of CA1 HP neurons positive for Tau7 at 4 (C), 14 (D), and 24 (E) months. The red rectangles are examples of the areas that fluorescence intensities were measured within individual neurons. F, Image illustrating the regions (in red) used for analysis of somatic tau intensity in the dentate gyrus. G-I, Enlargements represent the analyzed regions of 4-month-old (G), 14-month-old (H), and 24-month-old (I) rats. Scale bar, 20 µm.
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