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GIT2 acts as a potential keystone protein in functional hypothalamic networks associated with age-related phenotypic changes in rats.

Chadwick W, Martin B, Chapter MC, Park SS, Wang L, Daimon CM, Brenneman R, Maudsley S - PLoS ONE (2012)

Bottom Line: However, the proteomic effects of aging in regions of the brain vital for integrating energy balance and neuronal activity are not well understood.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.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.

View Article: PubMed Central - PubMed

Affiliation: Receptor Pharmacology Unit, Laboratory of Neuroscience, National Institute on Aging, National Institutes of Health, Biomedical Research Center, Baltimore, Maryland, United States of America.

ABSTRACT
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.

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Age-related proteomic alterations in the hypothalamus.(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.
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pone-0036975-g001: Age-related proteomic alterations in the hypothalamus.(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.

Mentions: Cytoplasmic hypothalamic extracts were prepared from young (Y, 2–3 months), middle-aged (M, 10–12 months), and old (O, 24–26 months) rats (n = 8 each). Individual lysate samples were taken from each animal and then pooled together for each age group. One dimensional gel separation was performed to control for any gross proteome differences between the three tissue pools (Fig. 1A). Gel separation indicated that after coomassie staining the pool inputs did not grossly differ in their global protein content. These samples were then prepared for Panorama® Cell Signaling antibody array hybridization by labeling with Cy-3 or Cy-5 fluorescent dyes (Fig. 1B, C). Relative protein expression between middle-aged (M) or old (O) versus young (Y) animals was assessed (in triplicate) using standardized dye-swapping controls as described previously [2]. Compared to young animals, there were 50 significantly differentially expressed proteins in middle aged animals, demonstrating an M/Y expression ratio of >1.5 (p<0.05) and 55 proteins with an M/Y ratio of <0.5 (p<0.05) (Fig. 1D:Table S1). Considerably more proteins demonstrated a significant change in expression between the old and the young animal comparison: 118 proteins demonstrated an O/Y expression ratio of >1.5 (p<0.05) and 30 proteins possessed an O/Y expression ratio of <0.5 (p<0.05) (Fig. 1E:Table S2). We chose six proteins identified in all the antibody array samples (demonstrating up, down, or no change in expression regulation: Myc, Akt, Pyk2, Map2, FAK, Cnp1) to validate the initial experiments using standard western blot procedures (Fig. 1F–K). Using the pooled hypothalamic samples (Y, M, O), we validated the expression trends for each of these proteins (Myc, Pyk2, FAK: up-regulated with advanced age; Akt, Map2: down-regulated with advanced age; Cnp1: unchanged with advanced age) seen with the Panorama® Cell Signaling array analysis (Tables S1, S2). In addition to standard western analysis of the input pooled hypothalamic samples, we also performed validatory western analysis on the individual animal samples (Fig. 1L–Q). Similarly to the pooled samples, we observed the following significant protein expression trends: Myc, Pyk2, FAK: up-regulated with advanced age; Akt, Map2: down-regulated with advanced age; Cnp: unchanged across age-span (Fig. 1L–Q).


GIT2 acts as a potential keystone protein in functional hypothalamic networks associated with age-related phenotypic changes in rats.

Chadwick W, Martin B, Chapter MC, Park SS, Wang L, Daimon CM, Brenneman R, Maudsley S - PLoS ONE (2012)

Age-related proteomic alterations in the hypothalamus.(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.
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Related In: Results  -  Collection

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pone-0036975-g001: Age-related proteomic alterations in the hypothalamus.(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.
Mentions: Cytoplasmic hypothalamic extracts were prepared from young (Y, 2–3 months), middle-aged (M, 10–12 months), and old (O, 24–26 months) rats (n = 8 each). Individual lysate samples were taken from each animal and then pooled together for each age group. One dimensional gel separation was performed to control for any gross proteome differences between the three tissue pools (Fig. 1A). Gel separation indicated that after coomassie staining the pool inputs did not grossly differ in their global protein content. These samples were then prepared for Panorama® Cell Signaling antibody array hybridization by labeling with Cy-3 or Cy-5 fluorescent dyes (Fig. 1B, C). Relative protein expression between middle-aged (M) or old (O) versus young (Y) animals was assessed (in triplicate) using standardized dye-swapping controls as described previously [2]. Compared to young animals, there were 50 significantly differentially expressed proteins in middle aged animals, demonstrating an M/Y expression ratio of >1.5 (p<0.05) and 55 proteins with an M/Y ratio of <0.5 (p<0.05) (Fig. 1D:Table S1). Considerably more proteins demonstrated a significant change in expression between the old and the young animal comparison: 118 proteins demonstrated an O/Y expression ratio of >1.5 (p<0.05) and 30 proteins possessed an O/Y expression ratio of <0.5 (p<0.05) (Fig. 1E:Table S2). We chose six proteins identified in all the antibody array samples (demonstrating up, down, or no change in expression regulation: Myc, Akt, Pyk2, Map2, FAK, Cnp1) to validate the initial experiments using standard western blot procedures (Fig. 1F–K). Using the pooled hypothalamic samples (Y, M, O), we validated the expression trends for each of these proteins (Myc, Pyk2, FAK: up-regulated with advanced age; Akt, Map2: down-regulated with advanced age; Cnp1: unchanged with advanced age) seen with the Panorama® Cell Signaling array analysis (Tables S1, S2). In addition to standard western analysis of the input pooled hypothalamic samples, we also performed validatory western analysis on the individual animal samples (Fig. 1L–Q). Similarly to the pooled samples, we observed the following significant protein expression trends: Myc, Pyk2, FAK: up-regulated with advanced age; Akt, Map2: down-regulated with advanced age; Cnp: unchanged across age-span (Fig. 1L–Q).

Bottom Line: However, the proteomic effects of aging in regions of the brain vital for integrating energy balance and neuronal activity are not well understood.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.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.

View Article: PubMed Central - PubMed

Affiliation: Receptor Pharmacology Unit, Laboratory of Neuroscience, National Institute on Aging, National Institutes of Health, Biomedical Research Center, Baltimore, Maryland, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus