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[Engadin]

Abstract

 

Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single‐gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age‐related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long‐lived animals with elevated Jun‐N‐terminal Kinase signaling. We find that these animals exhibit a delayed age‐related decline in protein turnover rates, as well as decreased steady‐state neuronal glucose‐6‐phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose‐6‐phosphate dehydrogenase (G6PD). Over‐expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long‐lived mutants.

 

 

1 INTRODUCTION

Studies in model organisms have generated significant insight into the genetic factors and environmental conditions that influence, cause, and prevent aging (Kennedy et al., 2014; Lopez‐Otin, Blasco, Partridge, Serrano, & Kroemer, 2013). Stress‐induced damage to biological macromolecules, altered energy metabolism, deregulation of processes ensuring cellular and tissue integrity, and loss of coordination of systemic physiological control mechanisms are among the drivers of the general loss of homeostasis observed in aging organisms (Riera & Dillin, 2015; Taylor & Dillin, 2011). Strikingly, single‐gene mutations have been identified that affect evolutionarily conserved signaling pathways and result in a coordinated delay of processes that contribute to the age‐related loss of homeostasis, thus significantly extending lifespan (Lopez‐Otin et al., 2013; Partridge, Alic, Bjedov, & Piper, 2011). However, the exact molecular mechanisms that link these mutations to their longevity‐promoting outcomes remain unclear. Characterizing the perturbation of interconnected downstream processes by longevity mutations is thus likely to provide significant insights into the aging process and to allow identifying novel longevity‐promoting interventions.

Changes in cellular metabolism and protein homeostasis (proteostasis) are likely central to the effects of many lifespan‐extending interventions. Mitochondrial activity and metabolic status are often changed by such interventions, reducing the production of reactive oxygen species (ROS) and other damaging metabolic by‐products (Avanesov et al., 2014; Houtkooper, Williams, & Auwerx, 2010; Riera & Dillin, 2015). A breakdown in proteostasis, in turn, is a central part of the normal aging process and is also evident in age‐related protein aggregation diseases of the brain, such as Alzheimer's disease (AD) (Labbadia & Morimoto, 2015; Taylor & Dillin, 2011). Improving proteostasis in model organisms, by increasing the expression of molecular chaperones, or by treating with compounds that reduce protein aggregates, can extend lifespan significantly (Alavez, Vantipalli, Zucker, Klang, & Lithgow, 2011; Taylor & Dillin, 2011; Tower, 2011). Similarly, increasing expression or function of the protein degradation machinery can significantly increase lifespan (Chondrogianni, Georgila, Kourtis, Tavernarakis, & Gonos, 2015; Juhasz, Erdi, Sass, & Neufeld, 2007; Vilchez et al., 2012), while reducing the rate of protein synthesis through depletion of translation initiation factors, ribosomal proteins, or ribosomal protein regulators increases lifespan in yeast, worms, and flies (Hansen et al., 2007; Steffen et al., 2008; Syntichaki, Troulinaki, & Tavernarakis, 2007; Wang, Cui, Jiang, & Xie, 2014). Lifespan can further be extended in all tested model organisms by inhibiting insulin‐IGF signaling (IIS) and Tor, a central nutrient sensor that influences protein homeostasis by a number of mechanisms (Kenyon, 2005; Lopez‐Otin et al., 2013; Partridge et al., 2011). Together, these studies point to metabolic perturbations and the loss of proteostasis as central drivers of deleterious aging phenotypes and age‐related diseases (Labbadia & Morimoto, 2015; Lopez‐Otin et al., 2013; Taylor & Dillin, 2011).

One of the most prominent proteostatic disruptions that are observed in aging cells and tissues is a global decrease in protein turnover rates. Age‐related increases in protein half‐lives have been documented in a number of species, including nematodes (Prasanna & Lane, 1979), rats (Lewis, Goldspink, Phillips, Merry, & Holehan, 1985), and humans (Young, Steffee, Pencharz, Winterer, & Scrimshaw, 1975) and may drive aberrant protein accumulation and post‐translational modification patterns (Rattan, 2010). These alterations can lead to a variety of irreversible protein damage, rendering cells dysfunctional and potentially accelerating the aging process. Reversing this age‐induced decline in protein turnover rates could be a potential downstream mechanism by which longevity‐promoting mutations extend lifespan. However, whether and how cellular proteostasis is influenced by such mutations is unclear, and the molecular mechanism(s) of the age‐associated decline in protein turnover are incompletely understood.

It further remains to be established how other metabolic, stress, and repair responses induced by longevity mutations are integrated with proteostatic changes to influence lifespan. Flies with increased Jun‐N‐terminal kinase (JNK) activity, for example, show a robust extension of lifespan that is mediated by the inhibition of insulin‐like peptide expression in insulin‐producing cells (Wang, Bohmann, & Jasper, 2003, 2005 ), by the induction of insulin resistance in peripheral tissues, as well as by JNK‐mediated improvement of cell‐autonomous protection and repair capabilities (Biteau, Karpac, Hwangbo, & Jasper, 2011). While increased JNK signaling activity thus induces metabolic changes and cytoprotective gene expression that are representative of IIS inhibition (Figure 1a), these changes may also be accompanied by the induction of proteostatic changes (Wu, Wang, & Bohmann, 2009; Xu, Das, Reilly, & Davis, 2011). How these different aspects of the response to JNK are integrated to achieve longevity remains unclear.

 

Figure 1

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Age‐related changes in protein turnover rates are reduced in heads of puc heterozygotes. (a) Model for JNK‐mediated regulation of metabolic homeostasis and lifespan extension. JNK‐mediated repression of insulin‐like peptide 2 (ILP2) expression in insulin‐producing cells (IPCs) of the fly brain results in systemic repression of insulin signaling. This, in turn, promotes FOXO activity and reduces Tor activity, promoting autophagy and limiting translation. JNK‐induced cytoprotective gene expression in the periphery contributes to longevity in JNK gain‐of‐function conditions. How such conditions influence changes in metabolic and protein homeostasis globally remains unclear. (b) Experimental approach to comprehensively characterize protein turnover rates in fly heads. Young (5‐day‐old) and old (35‐day‐old) flies were fed a sugar/yeast diet for 8 days in which 15N‐pre‐labeled yeast was the only nitrogen source. Proteins were isolated from whole heads and subjected to mass spectrometry. © Example for protein turnover analysis from mass spectrometry data. m/zdistribution of peptide P61851 derived from superoxide dismutase (SOD) in heads of young and old ry flies. Left panels: flies provided with an all 14N diet (14N yeast); Right panels: flies that were fed 15N‐labeled yeast for 8 days. Natural isotopic distribution of the peptide is shown in red. Distribution of 15N‐labeled peptide is shown in blue. The fraction labeled was calculated as described in Materials and Methods. (d) Distribution of protein half‐lives from heads of young (5 days) and old (35 days) ry or puc mutant flies. Note the shift toward longer protein half‐lives in old ry heads, but not in old puc mutant heads. (e) Comparison of protein half‐lives from heads of young (5 days) and old (35 days) ry or puc mutant flies. Each dot represents one protein. Note the deviation toward longer half‐lives (away from the blue diagonal with slope 1) in old ry heads but not in old puc heads. The blue lines indicate the theoretical identity line, and the red line indicates the best linear fit to the data. The box plots indicate the interquartile (box) and the complete (whiskers) range of the data excluding outliers (>2*SD). In the boxplots, the data have been limited to the subset shared between the compared sets. The white line and numbers indicate the median values. Comparisons and p value measurements were conducted by the Mann–Whitney U test.

 

 

Addressing these questions requires new approaches that comprehensively characterize the impact of longevity‐promoting interventions on metabolism and proteostasis in a complex organism. Highly sensitive and high‐throughput mass spectrometry technologies are beginning to allow such comprehensive assessments of protein turnover and metabolic flux and can be complemented by high‐throughput characterization of cellular transcriptomes. Here, we employ such technologies to characterize the metabolic and proteostatic effects of longevity‐promoting JNK gain‐of‐function conditions on Drosophila brains. Our results confirm that in wild‐type animals, protein turnover rates are globally reduced as a function of age, and we find that this is mitigated in long‐lived mutants for the JNK‐specific phosphatase Puckered (pucE69). Using untargeted metabolite profiling by LC‐MS/MS, flux analysis based on the 13C‐glucose uptake, and transcriptome profiling, we further find that JNK triggers a shift in neuronal carbon flux toward the pentose–phosphate pathway (PPP) by inducing the expression of glucose‐6‐phosphate dehydrogenase (G6PD), increasing NADPH production and reducing oxidative stress. Our data suggest that this metabolic reprogramming is responsible for the proteostatic and longevity effects of the pucE69mutation, as these phenotypes can be mimicked by increasing expression of G6PD in neurons. Our combined proteomic and metabolomic analysis suggests that JNK activation extends lifespan through metabolic reprogramming that promotes neuronal proteostasis.

 

 

More here: https://onlinelibrar...1111/acel.12849

 

 

 

 

 

 

Edited by Engadin, 03 March 2019 - 09:09 PM.