Loss of metabolic plasticity underlies metformin toxicity in aged Caenorhabditis elegans.

Current clinical trials are testing the life-extending benefits of the diabetes drug metformin in healthy individuals without diabetes. However, the metabolic response of a non-diabetic cohort to metformin treatment has not been studied. Here, we show in C. elegans and human primary cells that metformin shortens lifespan when provided in late life, contrary to its positive effects in young organisms. We find that metformin exacerbates ageing-associated mitochondrial dysfunction, causing respiratory failure. Age-related failure to induce glycolysis and activate the dietary-restriction-like mobilization of lipid reserves in response to metformin result in lethal ATP exhaustion in metformin-treated aged worms and late-passage human cells, which can be rescued by ectopic stabilization of cellular ATP content. Metformin toxicity is alleviated in worms harbouring disruptions in insulin-receptor signalling, which show enhanced resilience to mitochondrial distortions at old age. Together, our data show that metformin induces deleterious changes of conserved metabolic pathways in late life, which could bring into question its benefits for older individuals without diabetes.

It is not all about regeneration: Planarians striking power to stand starvation.

All living forms, prokaryotes as eukaryotes, have some means of adaptation to food scarcity, which extends the survival chances under extreme environmental conditions. Nowadays we know that dietary interventions, including fasting, extends lifespan of many organisms and can also protect against age-related diseases including in humans. Therefore, the capacity of adapting to periods of food scarcity may have evolved billions of years ago not only to allow immediate organismal survival but also to be able to extend organismal lifespan or at least to lead to a healthier remaining lifespan. Planarians have been the center of attention since more than two centuries because of their astonishing power of full body regeneration that relies on a large amount of adult stem cells or neoblasts. However, they also present an often-overlooked characteristic. They are able to stand long time starvation. Planarians have adapted to periods of fasting by shrinking or degrowing. Here we will review the published data about starvation in planarians and conclude with the possibility of starvation being one of the processes that rejuvenate the planarian, thus explaining the historical notion of non-ageing planarians.

A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair.

Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis.

ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines.

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3ß blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator.

E2F1 regulates cellular growth by mTORC1 signaling.

During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation.

Study of the in vivo phosphorylation of E2F1 on Ser403.

Multiple E2F1 phosphorylation sites have been described as targets of different kinases, yet their in vivo implication is uncertain. We previously reported that GSK3beta is able to phosphorylate E2F1 in vitro at Ser403 and Ser433. Recently, it has been shown that both residues are also direct targets of p38 MAP kinase. In order to determine whether Ser403 phosphorylation occurs in vivo and to elucidate its role in E2F1 transcription activity, we developed a phospho-E2F1(Ser403) antibody for use in in vivo detection studies. Our results demonstrate that endogenous E2F1 is phosphorylated in vivo on Ser403, however neither GSK3beta nor p38 MAP kinase are responsible for this event. E2F1 phosphorylation on Ser403 is induced after treatment with doxorubicin in a dose response manner. The transcriptional response of E2F1 to doxorubicin is lower in an E2F1 Ser/Ala403 mutated construct relative to the wild type, suggesting a role for Ser403 phosphorylation in DNA damage conditions. Comparative study between the expression of the bcl2 gene family induced by the wild type and E2F1 Ser/Ala403 mutant revealed a statistically different pattern between both conditions. These results suggest that phosphorylation of Ser403 could influence the selection and regulation of E2F1 target genes.

Apoptotic action of E2F1 requires glycogen synthase kinase 3-beta activity in PC12 cells.

Both E2F1 and GSK3beta have been described as essential targets in neuronal apoptosis. Previous studies have demonstrated that GSK3beta binds to E2F1 in vivo. We wanted to investigate whether these proteins could share a common apoptotic signal pathway in neuronal cells. With this intention, we developed a PC12 ER-E2F1 stable cell line in which E2F1 activity was dependent on the presence of 4-hydroxitamoxifen. E2F1 activation produced apoptosis in naive and post-mitotic cells; serum and nerve growth factor respectively protected them from E2F1 apoptotic stimuli. The presence of specific GSK3beta inhibitors SB216763 and LiCl completely protected cells from apoptosis induced by E2F1 activation. In addition, knocked down GSK3beta experiments by small interference RNAs have demonstrated that a reduction of GSK3beta protein levels can lower the apoptotic effect of E2F1. Finally, we demonstrated that the apoptotic effect of E2F1 is not due to the regulation of GSK3beta activity, and that the inhibitory effect of GSK3beta inhibitor SB216763 on E2F1 induced apoptosis could be due to an alteration in the E2F1-regulated transcription gene pattern. In summary, we have demonstrated that the apoptotic action of E2F1 requires GSK3beta activity.