GIT2 Acts as a Potential Keystone Protein in Functional Hypothalamic Networks Associated with Age-Related Phenotypic Changes in Rats

The aging process affects every tissue in the body and represents one of the most complicated and highly integrated inevitable physiological entities. The maintenance of good health during the aging process likely relies upon the coherent regulation of hormonal and neuronal communication between the central nervous system and the periphery. Evidence has demonstrated that the optimal regulation of energy usage in both these systems facilitates healthy aging. However, the proteomic effects of aging in regions of the brain vital for integrating energy balance and neuronal activity are not well understood. The hypothalamus is one of the main structures in the body responsible for sustaining an efficient interaction between energy balance and neurological activity. Therefore, a greater understanding of the effects of aging in the hypothalamus may reveal important aspects of overall organismal aging and may potentially reveal the most crucial protein factors supporting this vital signaling integration. In this study, we examined alterations in protein expression in the hypothalami of young, middle-aged, and old rats. Using novel combinatorial bioinformatics analyses, we were able to gain a better understanding of the proteomic and phenotypic changes that occur during the aging process and have potentially identified the G protein-coupled receptor/cytoskeletal-associated protein GIT2 as a vital integrator and modulator of the normal aging process

Correlation of GIT2 and GIT2s expression with metabolic phenotype changes in aging rats.

<p>(A) Age-dependent changes in mean body weight for the multiple age ranges of rats. (B) Fasting blood glucose measurements for the multiple age ranges of rats. (C) Fasting plasma insulin measurements for the multiple age ranges of rats. (D) Fasting plasma leptin measurements for the multiple age ranges of rats. (E) Fasting plasma adiponectin measurements for the multiple age ranges of rats. (F) GIT2 and GIT2s age-dependent expression in young (Y), middle-aged (M), and old rat hypothalamic extracts. The associated histograms depict the mean ± SEM of the GIT2 and GIT2s expression in middle-aged (grey bars) and old (black bars) animals relative to the young (white bars) samples. (G) GIT2 and GIT2s age-dependent expression in young (Y), middle-aged (M), and old rat whole pancreatic extracts. The associated histograms depict the mean ± SEM of the GIT2 and GIT2s expression in middle-aged (grey bars) and old (black bars) animals relative to the young (white bars) samples. (H) GIT2 and GIT2s age-dependent expression in young (Y), middle-aged (M), and old rat whole liver extracts. The associated histograms depict the mean ± SEM of the GIT2 and GIT2s expression in middle-aged (grey bars) and old (black bars) animals relative to the young (white bars) samples. (I) GIT2 and GIT2s age-dependent expression in young (Y), middle-aged (M), and old rat hind-limb skeletal muscle extracts. The associated histograms depict the mean ± SEM of the GIT2 and GIT2s expression in middle-aged (grey bars) and old (black bars) animals relative to the young (white bars) samples. (J) GIT2 and GIT2s age-dependent expression in young (Y), middle-aged (M), and old rat somatic adipose tissue extracts. The associated histograms depict the mean ± SEM of the GIT2 and GIT2s expression in middle-aged (grey bars) and old (black bars) animals relative to the young (white bars) samples. Statistical significance is as follows: * = p<0.05, ** = p<0.01, *** = p<0.001.</p

KEGG signaling pathway analysis of aging-related hypothalamic proteins.

<p>Proteins significantly regulated in middle (M) or old (O) aged animals compared to young (Y) animals were used as input data for KEGG signaling pathway population analysis. (A) Venn diagram analysis of middle-aged (grey line) and old-aged (black line) significantly-regulated KEGG pathways demonstrated 27 common KEGG terms between old and middle-aged tissues. The common (27) significantly populated pathways for middle-aged (grey bars) and old aged (black bars) animals were then rationally clustered into subgroups focused upon disease pathology (B), neurophysiological activity (C), and intermediary cellular signaling activity (D). For each significantly-populated KEGG pathway a ‘hybrid’ score was generated which represents the −log₁₀ of the enrichment probability multiplied with the relative enrichment factor compared to the background proteomic expression.</p

Age-related proteomic alterations in the hypothalamus.

<p>(A) Coomassie staining of pooled input hypothalami for Cy-dye labeling and hybridization with Cell Signaling antibody array: Y–young animal pool; M–middle-aged animal pool; O–old animal pool. (B) Prototypic single channel and merge images from scanned Panorama® Cell Signaling Antibody array chips. Specific antibody species are printed in duplicate across the chip. (C) Prototypic examples of protein results for factors up-regulated, down-regulated, or unchanged in either Cy3 or Cy5 channels are indicated. Positive and negative hybridization controls from the chips are also indicated. (D) Protein expression ratios (<0.5 or >1.5 ratio: middle versus young) for proteins in middle aged versus young hypothalami. Datapoints plotted represent the mean ± standard error of mean (SEM) from three separate experimental hybridizations which included Cys-Cy5 dye swaps for the samples. (E) Protein expression ratios (<0.5 or >1.5 ratio: old versus young) for proteins in old aged versus young hypothalami. Datapoints plotted represent the mean ± SEM from three separate experimental hybridizations which included Cys-Cy5 dye swaps for the samples. (F–K) Western blot validation of specifically identified proteins, from Panorama® array analysis, and their age-dependent expression trends (Y-young pool, M-middle aged pool, O-old pool). Proteins validated from pooled animal input were Myc (F), Akt-1 (G), Pyk2 (H), Map2 (I), FAK (J), and Cnp-1 (K). Data presented represents mean ± SEM from three separate blots. Statistical significance was assessed using a Student's t-test with GraphPad Prism: * = p<0.05; ** = p<0.01; *** = p<0.001. (L–Q) Expression patterns for specific proteins were also validated for each animal used as input for the Y (white circle), M (grey square), or O (black triangle) hypothalamic pools. Proteins validated from individual animal inputs were Myc (L), Akt-1 (M), Pyk2 (N), Map2 (O), FAK (P) and Cnp-1 (Q). Data on the histograms are represented as mean ± SEM from the multiple animals.</p

Age-dependent expression profile of GIT2 in central nervous system tissues.

<p>(A) Expression profiles across three randomly chosen hypothalamic samples (Young, Y₁-Y₂-Y₃; Middle, M₁-M₂-M₃; Old, O₁-O₂-O₃) for ERK1/2, GRIT, GIT2, GIT2short (GIT2s), GIT1, β-PIX, and PAK1. The loading protein input control with coomassie staining of the gel is also indicated below. (B) Quantification (mean ± SEM) of age-dependent alterations in protein expression for middle aged (grey bars) or old (black bars) animals compared to the young controls (white bars). (C) Brain region-specific alterations of GIT2 and GIT2s in young (Y), middle (M), and old (O) age animals. Quantification of multi-brain region GIT2 (D) and GIT2s (E) expression across the experimental age-span (n = 10). Multiple brain region expression levels relative to hypothalamus of GIT2 in young (F), middle-aged (G), and old animals (H). Multiple brain region expression levels, relative to hypothalamus of GIT2s in young (I), middle-aged (J), and old animals (K). Statistical significance is as follows: * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Latent semantic indexing correlations of KEGG signaling pathways terms with proteins.

<p>(A) Latent semantic indexing (LSI) interrogation matrix between input significantly-regulated KEGG signaling pathway terms. Colored blocks represent the individual LSI implicit correlation of the specific protein (vertically organized on left of heatmap: 1–2524 – see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036975#pone.0036975.s024" target="_blank">Table S20</a>) with the respective KEGG term (1-Regulation of actin cytoskeleton, 2-Chemokine signaling, 3-Alzheimer's disease, 4-Focal adhesion, 5-MAPK signaling, 6-Gap junction, 7-GnRH signaling, 8-Long term potentiation, 9-Notch signaling, 10-VEGF signaling, 11-p53 signaling, 12-Calcium signaling). The number of KEGG signaling pathway correlations for each protein is indicated by the color of the respective heatmap blocks (9 correlations-red; 8 correlations-orange; 7 correlations-yellow; 6 correlations-green; 5 correlations-light blue; 4 correlations-dark blue; 3 correlations-purple; 2 correlations-grey). (B) Mean ± SEM for the total implicitly-correlating proteins for each of the 12 input KEGG signaling pathways. (C) Box and whisker plot with 1–99% statistical cut-offs (GraphPad Prism) of the number of specific correlations to KEGG pathways each protein possessed. Twelve proteins demonstrated a statistically-significantly greater number of KEGG pathway correlations compared to the total protein mean number of correlations (*** = p<0.001). (D) Expanded heatmap identification of specific proteins possessing a significantly greater number of KEGG pathway correlations compared to the mean number of KEGG pathway correlations for all implicit proteins. (E) Mean ± SEM of LSI correlation scores (across all 9 correlations) for Grit and GIT2.</p

Age-dependent protein expression changes in the rat hypothalamus.

<p>(A) Venn diagram analysis of significantly-regulated (p<0.05) proteins in the middle versus young (grey circle) and the old versus young (black circle) antibody array analyses. Proteins uniquely regulated in either middle age (22) or old age (64) animals are subsequently broken down into up-regulated (red numbers) or down-regulated (green numbers) groups. The 84 commonly-regulated proteins were further dissected into different regulatory-behavior groups represented in panels B–E. For each regulation-behavior group an exemplary protein is verified from the animal hypothalamic pools (Y–young, M–middle aged, O–old). (B) Proteins up-regulated in both M (grey bars) and O (black bars) hypothalami compared to Y animals, with O ratio >M ratio. The associated validation was performed using western blot for caspase 3 (Casp3). (C) Proteins up-regulated in both M (grey bars) and O (black bars) hypothalami compared to Y animals, with M ratio >O ratio. The associated validation was performed using western blot for Ran. (D) Proteins down-regulated in both M (grey bars) and O (black bars) hypothalami compared to Y animals, with M ratio >O ratio and O ratio >M ratio. The associated validation was performed using western blot for vinculin (Vcl). (E) Proteins differentially regulated between M or O timepoints relative to Y (up-regulated at M and down-regulated at O (verified using junction plakoglobin-Jup), or down-regulated at M and up-regulated at O (verified using nitric oxide synthase 1-Nos)). For each verification, data on each histogram is represented as mean ± SEM from the multiple animal pools. Statistical significance is as follows: * = p<0.05, ** = p<0.01, *** = p<0.001.</p