Xrp1 governs the stress response program to spliceosome dysfunction.

Co-transcriptional processing of nascent pre-mRNAs by the spliceosome is vital to regulating gene expression and maintaining genome integrity. Here, we show that the deficiency of functional U5 small nuclear ribonucleoprotein particles (snRNPs) in Drosophila imaginal cells causes extensive transcriptome remodeling and accumulation of highly mutagenic R-loops, triggering a robust stress response and cell cycle arrest. Despite compromised proliferative capacity, the U5 snRNP-deficient cells increased protein translation and cell size, causing intra-organ growth disbalance before being gradually eliminated via apoptosis. We identify the Xrp1-Irbp18 heterodimer as the primary driver of transcriptional and cellular stress program downstream of U5 snRNP malfunction. Knockdown of Xrp1 or Irbp18 in U5 snRNP-deficient cells attenuated JNK and p53 activity, restored normal cell cycle progression and growth, and inhibited cell death. Reducing Xrp1-Irbp18, however, did not rescue the splicing defects, highlighting the requirement of accurate splicing for cellular and tissue homeostasis. Our work provides novel insights into the crosstalk between splicing and the DNA damage response and defines the Xrp1-Irbp18 heterodimer as a critical sensor of spliceosome malfunction and mediator of the stress-induced cellular senescence program.

The removal of introns and the joining of exons into mature mRNA by the spliceosome is crucial in regulating gene expression, simultaneously safeguarding genome integrity and enhancing proteome diversity in multicellular organisms. Spliceosome dysfunction is thus associated with various diseases and organismal aging. Our study describes the cascade of events in response to spliceosome dysfunction. We identified two transcription factors as drivers of a stress response program triggered by spliceosome dysfunction, which dramatically remodel gene expression to protect tissue integrity and induce a senescent-like state in damaged cells prior to their inevitable elimination. Together, we highlight the indispensable role of spliceosomes in maintaining homeostasis and implicate spliceosome dysfunction in senescent cell accumulation associated with the pathomechanisms of spliceopathies and aging.

Ageing-associated changes in transcriptional elongation influence longevity.

Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1-4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin-IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures.

FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity.

Proteostasis collapses during aging resulting, among other things, in the accumulation of damaged and aggregated proteins. The proteasome is the main cellular proteolytic system and plays a fundamental role in the maintenance of protein homeostasis. Our previous work has demonstrated that senescence and aging are related to a decline in proteasome content and activities, while its activation extends lifespan in vitro and in vivo in various species. However, the mechanisms underlying this age-related decline of proteasome function and the down-regulation in expression of its subunits remain largely unclear. Here, we demonstrate that the Forkhead box-O1 (FoxO1) transcription factor directly regulates the expression of a 20S proteasome catalytic subunit and, hence, proteasome activity. Specifically, we demonstrate that knockout of FoxO1, but not of FoxO3, in mice severely impairs proteasome activity in several tissues, while depletion of IRS1 enhances proteasome function. Importantly, we show that FoxO1 directly binds on the promoter region of the rate-limiting catalytic ß5 proteasome subunit to regulate its expression. In summary, this study reveals the direct role of FoxO factors in the regulation of proteasome function and provides new insight into how FoxOs affect proteostasis and, in turn, longevity.

Longevity in response to lowered insulin signaling requires glycine N-methyltransferase-dependent spermidine production.

Reduced insulin/IGF signaling (IIS) extends lifespan in multiple organisms. Different processes in different tissues mediate this lifespan extension, with a set of interplays that remain unclear. We here show that, in Drosophila, reduced IIS activity modulates methionine metabolism, through tissue-specific regulation of glycine N-methyltransferase (Gnmt), and that this regulation is required for full IIS-mediated longevity. Furthermore, fat body-specific expression of Gnmt was sufficient to extend lifespan. Targeted metabolomics showed that reducing IIS activity led to a Gnmt-dependent increase in spermidine levels. We also show that both spermidine treatment and reduced IIS activity are sufficient to extend the lifespan of Drosophila, but only in the presence of Gnmt. This extension of lifespan was associated with increased levels of autophagy. Finally, we found that increased expression of Gnmt occurs in the liver of liver-specific IRS1 KO mice and is thus an evolutionarily conserved response to reduced IIS. The discovery of Gnmt and spermidine as tissue-specific modulators of IIS-mediated longevity may aid in developing future therapeutic treatments to ameliorate aging and prevent disease.

A triple drug combination targeting components of the nutrient-sensing network maximizes longevity.

Increasing life expectancy is causing the prevalence of age-related diseases to rise, and there is an urgent need for new strategies to improve health at older ages. Reduced activity of insulin/insulin-like growth factor signaling (IIS) and mechanistic target of rapamycin (mTOR) nutrient-sensing signaling network can extend lifespan and improve health during aging in diverse organisms. However, the extensive feedback in this network and adverse side effects of inhibition imply that simultaneous targeting of specific effectors in the network may most effectively combat the effects of aging. We show that the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib, the mTOR complex 1 (mTORC1) inhibitor rapamycin, and the glycogen synthase kinase-3 (GSK-3) inhibitor lithium act additively to increase longevity in Drosophila Remarkably, the triple drug combination increased lifespan by 48%. Furthermore, the combination of lithium with rapamycin cancelled the latter's effects on lipid metabolism. In conclusion, a polypharmacology approach of combining established, prolongevity drug inhibitors of specific nodes may be the most effective way to target the nutrient-sensing network to improve late-life health.

A proteomic atlas of insulin signalling reveals tissue-specific mechanisms of longevity assurance.

Lowered activity of the insulin/IGF signalling (IIS) network can ameliorate the effects of ageing in laboratory animals and, possibly, humans. Although transcriptome remodelling in long-lived IIS mutants has been extensively documented, the causal mechanisms contributing to extended lifespan, particularly in specific tissues, remain unclear. We have characterized the proteomes of four key insulin-sensitive tissues in a long-lived Drosophila IIS mutant and control, and detected 44% of the predicted proteome (6,085 proteins). Expression of ribosome-associated proteins in the fat body was reduced in the mutant, with a corresponding, tissue-specific reduction in translation. Expression of mitochondrial electron transport chain proteins in fat body was increased, leading to increased respiration, which was necessary for IIS-mediated lifespan extension, and alone sufficient to mediate it. Proteasomal subunits showed altered expression in IIS mutant gut, and gut-specific over-expression of the RPN6 proteasomal subunit, was sufficient to increase proteasomal activity and extend lifespan, whilst inhibition of proteasome activity abolished IIS-mediated longevity. Our study thus uncovered strikingly tissue-specific responses of cellular processes to lowered IIS acting in concert to ameliorate ageing.

Changes of mitochondrial ultrastructure and function during ageing in mice and Drosophila.

Ageing is a progressive decline of intrinsic physiological functions. We examined the impact of ageing on the ultrastructure and function of mitochondria in mouse and fruit flies (Drosophila melanogaster) by electron cryo-tomography and respirometry. We discovered distinct age-related changes in both model organisms. Mitochondrial function and ultrastructure are maintained in mouse heart, whereas subpopulations of mitochondria from mouse liver show age-related changes in membrane morphology. Subpopulations of mitochondria from young and old mouse kidney resemble those described for apoptosis. In aged flies, respiratory activity is compromised and the production of peroxide radicals is increased. In about 50% of mitochondria from old flies, the inner membrane organization breaks down. This establishes a clear link between inner membrane architecture and functional decline. Mitochondria were affected by ageing to very different extents, depending on the organism and possibly on the degree to which tissues within the same organism are protected against mitochondrial damage.

Reduced insulin/insulin-like growth factor signaling decreases translation in Drosophila and mice.

Down-regulation of insulin/insulin-like growth factor signaling (IIS) can increase lifespan in C. elegans, Drosophila and mice. In C. elegans, reduced IIS results in down-regulation of translation, which itself can extend lifespan. However, the effect of reduced IIS on translation has yet to be determined in other multicellular organisms. Using two long-lived IIS models, namely Drosophila lacking three insulin-like peptides (dilp2-3,5(-/-)) and mice lacking insulin receptor substrate 1 (Irs1(-/-)), and two independent translation assays, polysome profiling and radiolabeled amino acid incorporation, we show that reduced IIS lowers translation in these organisms. In Drosophila, reduced IIS decreased polysome levels in fat body and gut, but reduced the rate of protein synthesis only in the fat body. Reduced IIS in mice decreased protein synthesis rate only in skeletal muscle, without reducing polysomes in any tissue. This lowered translation in muscle was independent of Irs1 loss in the muscle itself, but a secondary effect of Irs1 loss in the liver. In conclusion, down-regulation of translation is an evolutionarily conserved response to reduced IIS, but the tissues in which it occurs can vary between organisms. Furthermore, the mechanisms underlying lowered translation may differ in mice, possibly associated with the complexity of the regulatory processes.

Lithium Promotes Longevity through GSK3/NRF2-Dependent Hormesis.

The quest to extend healthspan via pharmacological means is becoming increasingly urgent, both from a health and economic perspective. Here we show that lithium, a drug approved for human use, promotes longevity and healthspan. We demonstrate that lithium extends lifespan in female and male Drosophila, when administered throughout adulthood or only later in life. The life-extending mechanism involves the inhibition of glycogen synthase kinase-3 (GSK-3) and activation of the transcription factor nuclear factor erythroid 2-related factor (NRF-2). Combining genetic loss of the NRF-2 repressor Kelch-like ECH-associated protein 1 (Keap1) with lithium treatment revealed that high levels of NRF-2 activation conferred stress resistance, while low levels additionally promoted longevity. The discovery of GSK-3 as a therapeutic target for aging will likely lead to more effective treatments that can modulate mammalian aging and further improve health in later life.

Drosophila melanogaster LRPPRC2 is involved in coordination of mitochondrial translation.

Members of the pentatricopeptide repeat domain (PPR) protein family bind RNA and are important for post-transcriptional control of organelle gene expression in unicellular eukaryotes, metazoans and plants. They also have a role in human pathology, as mutations in the leucine-rich PPR-containing (LRPPRC) gene cause severe neurodegeneration. We have previously shown that the mammalian LRPPRC protein and its Drosophila melanogaster homolog DmLRPPRC1 (also known as bicoid stability factor) are necessary for mitochondrial translation by controlling stability and polyadenylation of mRNAs. We here report characterization of DmLRPPRC2, a second fruit fly homolog of LRPPRC, and show that it has a predominant mitochondrial localization and interacts with a stem-loop interacting RNA binding protein (DmSLIRP2). Ubiquitous downregulation of DmLrpprc2 expression causes respiratory chain dysfunction, developmental delay and shortened lifespan. Unexpectedly, decreased DmLRPPRC2 expression does not globally affect steady-state levels or polyadenylation of mitochondrial transcripts. However, some mitochondrial transcripts abnormally associate with the mitochondrial ribosomes and some products are dramatically overproduced and other ones decreased, which, in turn, results in severe deficiency of respiratory chain complexes. The function of DmLRPPRC2 thus seems to be to ensure that mitochondrial transcripts are presented to the mitochondrial ribosomes in an orderly fashion to avoid poorly coordinated translation.

Lithium suppresses Aß pathology by inhibiting translation in an adult Drosophila model of Alzheimer's disease.

The greatest risk factor for Alzheimer's disease (AD) is age, and changes in the ageing nervous system are likely contributors to AD pathology. Amyloid beta (Aß) accumulation, which occurs as a result of the amyloidogenic processing of amyloid precursor protein (APP), is thought to initiate the pathogenesis of AD, eventually leading to neuronal cell death. Previously, we developed an adult-onset Drosophila model of AD. Mutant Aß42 accumulation led to increased mortality and neuronal dysfunction in the adult flies. Furthermore, we showed that lithium reduced Aß42 protein, but not mRNA, and was able to rescue Aß42-induced toxicity. In the current study, we investigated the mechanism/s by which lithium modulates Aß42 protein levels and Aß42 induced toxicity in the fly model. We found that lithium caused a reduction in protein synthesis in Drosophila and hence the level of Aß42. At both the low and high doses tested, lithium rescued the locomotory defects induced by Aß42, but it rescued lifespan only at lower doses, suggesting that long-term, high-dose lithium treatment may have induced toxicity. Lithium also down-regulated translation in the fission yeast Schizosaccharomyces pombe associated with increased chronological lifespan. Our data highlight a role for lithium and reduced protein synthesis as potential therapeutic targets for AD pathogenesis.

Lowered insulin signalling ameliorates age-related sleep fragmentation in Drosophila.

Sleep fragmentation, particularly reduced and interrupted night sleep, impairs the quality of life of older people. Strikingly similar declines in sleep quality are seen during ageing in laboratory animals, including the fruit fly Drosophila. We investigated whether reduced activity of the nutrient- and stress-sensing insulin/insulin-like growth factor (IIS)/TOR signalling network, which ameliorates ageing in diverse organisms, could rescue the sleep fragmentation of ageing Drosophila. Lowered IIS/TOR network activity improved sleep quality, with increased night sleep and day activity and reduced sleep fragmentation. Reduced TOR activity, even when started for the first time late in life, improved sleep quality. The effects of reduced IIS/TOR network activity on day and night phenotypes were mediated through distinct mechanisms: Day activity was induced by adipokinetic hormone, dFOXO, and enhanced octopaminergic signalling. In contrast, night sleep duration and consolidation were dependent on reduced S6K and dopaminergic signalling. Our findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans.

Translating translation: regulated protein translation as a biomedical intervention.

Altering the cellular response to internal and external stressors is essential for survival, hence the process of translation is exquisitely regulated to rapidly change the proteomic profile upon physiological challenges. We recently reported that genetic and pharmacological manipulation of translation may be beneficial in the treatment of Parkinson disease (PD). Using two Drosophila models of PD, we showed that altering the regulation of protein translation is sufficient to ameliorate the phenotypes of these models, including neurodegeneration, mitochondrial defects and behavioral deficits. Previous studies implicating translation regulation in lifespan extension further implicates this as an important mechanism that can mediate cell protective pathways, not just for age-related diseases such as PD, but also of aging itself. As such, translation regulation represents a convergent target for therapeutic interventions. Here we highlight the therapeutic potential of translation regulation in disease and describe how determining profiles of protein synthesis may help in the fight for disease prevention and healthy aging.

Rapamycin activation of 4E-BP prevents parkinsonian dopaminergic neuron loss.

Mutations in PINK1 and PARK2 cause autosomal recessive parkinsonism, a neurodegenerative disorder that is characterized by the loss of dopaminergic neurons. To discover potential therapeutic pathways, we identified factors that genetically interact with Drosophila park and Pink1. We found that overexpression of the translation inhibitor Thor (4E-BP) can suppress all of the pathologic phenotypes, including degeneration of dopaminergic neurons in Drosophila. 4E-BP is activated in vivo by the TOR inhibitor rapamycin, which could potently suppress pathology in Pink1 and park mutants. Rapamycin also ameliorated mitochondrial defects in cells from individuals with PARK2 mutations. Recently, 4E-BP was shown to be inhibited by the most common cause of parkinsonism, dominant mutations in LRRK2. We also found that loss of the Drosophila LRRK2 homolog activated 4E-BP and was also able to suppress Pink1 and park pathology. Thus, in conjunction with recent findings, our results suggest that pharmacologic stimulation of 4E-BP activity may represent a viable therapeutic approach for multiple forms of parkinsonism.

Dietary regulation of hypodermal polyploidization in C. elegans.

BACKGROUND: Dietary restriction (DR) results in increased longevity, reduced fecundity and reduced growth in many organisms. Though many studies have examined the effects of DR on longevity and fecundity, few have investigated the effects on growth. RESULTS: Here we use Caenorhabditis elegans to determine the mechanisms that regulate growth under DR. We show that rather than a reduction in cell number, decreased growth in wild type C. elegans under DR is correlated with lower levels of hypodermal polyploidization. We also show that mutants lacking wild type sensory ciliated neurons are small, exhibit hypo-polyploidization and more importantly, when grown under DR, reduce their levels of endoreduplication to a lesser extent than wild type, suggesting that these neurons are required for the regulation of hypodermal polyploidization in response to DR. Similarly, we also show that the cGMP-dependent protein kinase EGL-4 and the SMA/MAB signalling pathway regulate polyploidization under DR. CONCLUSION: We show C. elegans is capable of actively responding to food levels to regulate adult ploidy. We suggest this response is dependent on the SMA/MAB signalling pathway.