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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-dependent expression profile of GIT2 in central nervous system tissues.(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.
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pone-0036975-g005: Age-dependent expression profile of GIT2 in central nervous system tissues.(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.

Mentions: From our combinatorial bioinformatic investigation of predicted age-dependent protein expression in the hypothalamus, we decided to assess the age-dependent expression of GIT2 using standardized techniques. We assessed protein expression with specific western blots in three randomly-chosen young (Y1, Y2, Y3), middle-aged (M1, M2, M3), and old rat hypothalami (O1, O2, O3: Fig. 5A). We found that with similar levels of loaded protein (10 µg), the ERK1/2 expression profile was unchanged with age but the expression profile for Grit and GIT2 was strongly age-dependent (Fig. 5A–B). The age-dependent elevation of GIT2 was extremely profound while that of Grit was less strong. GIT2 often demonstrates co-expression with a shorter isoform, termed GIT2-short (GIT2s). Similar to the age-dependent increase in GIT2 hypothalamic expression, we also found a strong elevation of GIT2s with age (Fig. 5A). The GIT family of proteins consist of GIT1 and GIT2, as well as the smaller isoforms of GIT2 including GIT2s [22]. Proteins belonging to the GIT family were first identified as ADP-ribosylation factor GTPase-activating proteins. GIT proteins, and GIT1 especially, associate with signaling factors, p21-activated kinase (PAK) and PAK-interacting exchange factor (PIX), and the monomeric G proteins that control cytoskeletal remodeling, e.g. Rac and Cdc42, which regulate cell structure and movement [22]. However, in contrast to GIT2 and GIT2s, the levels of GIT1, β-PIX, or PAK1 did not demonstrate a significant elevation with age in the hypothalamus (Fig. 5A–B), suggesting a strong age-dependent hypothalamic functionality for GIT2. As we have seen that protein expression of GIT2 is altered during the aging process, we also investigated whether alterations in post-translational modification of GIT2 are also sensitive to aging. GIT2 functional activity has been demonstrated to be associated with its tyrosine phosphorylation [23]. We assessed the tyrosine phosphorylation status of GIT2 in normalized (for GIT2) hypothalamic lysates collected from rats of the different ages. We found a trend for age-dependent elevation of the phosphotyrosine content in hypothalamic GIT2 (Fig. S3). This increase in phosphotyrosine content may indeed be associated with the age-related elevation in kinases linked to GIT2 phosphorylation such as FAK (Fig. 1). We next assessed whether there were age-dependent changes in GIT2 and GIT2s expression across other regions of the central nervous system outside our primary hypothalamic locus.


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-dependent expression profile of GIT2 in central nervous system tissues.(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351446&req=5

pone-0036975-g005: Age-dependent expression profile of GIT2 in central nervous system tissues.(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.
Mentions: From our combinatorial bioinformatic investigation of predicted age-dependent protein expression in the hypothalamus, we decided to assess the age-dependent expression of GIT2 using standardized techniques. We assessed protein expression with specific western blots in three randomly-chosen young (Y1, Y2, Y3), middle-aged (M1, M2, M3), and old rat hypothalami (O1, O2, O3: Fig. 5A). We found that with similar levels of loaded protein (10 µg), the ERK1/2 expression profile was unchanged with age but the expression profile for Grit and GIT2 was strongly age-dependent (Fig. 5A–B). The age-dependent elevation of GIT2 was extremely profound while that of Grit was less strong. GIT2 often demonstrates co-expression with a shorter isoform, termed GIT2-short (GIT2s). Similar to the age-dependent increase in GIT2 hypothalamic expression, we also found a strong elevation of GIT2s with age (Fig. 5A). The GIT family of proteins consist of GIT1 and GIT2, as well as the smaller isoforms of GIT2 including GIT2s [22]. Proteins belonging to the GIT family were first identified as ADP-ribosylation factor GTPase-activating proteins. GIT proteins, and GIT1 especially, associate with signaling factors, p21-activated kinase (PAK) and PAK-interacting exchange factor (PIX), and the monomeric G proteins that control cytoskeletal remodeling, e.g. Rac and Cdc42, which regulate cell structure and movement [22]. However, in contrast to GIT2 and GIT2s, the levels of GIT1, β-PIX, or PAK1 did not demonstrate a significant elevation with age in the hypothalamus (Fig. 5A–B), suggesting a strong age-dependent hypothalamic functionality for GIT2. As we have seen that protein expression of GIT2 is altered during the aging process, we also investigated whether alterations in post-translational modification of GIT2 are also sensitive to aging. GIT2 functional activity has been demonstrated to be associated with its tyrosine phosphorylation [23]. We assessed the tyrosine phosphorylation status of GIT2 in normalized (for GIT2) hypothalamic lysates collected from rats of the different ages. We found a trend for age-dependent elevation of the phosphotyrosine content in hypothalamic GIT2 (Fig. S3). This increase in phosphotyrosine content may indeed be associated with the age-related elevation in kinases linked to GIT2 phosphorylation such as FAK (Fig. 1). We next assessed whether there were age-dependent changes in GIT2 and GIT2s expression across other regions of the central nervous system outside our primary hypothalamic locus.

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