Nutrient sensing and the Metabolic control of cell growth / Intégration des signaux nutritifs et contrôle métabolique de la croissance cellulaire

Séminaire organisé par Mario Pende (Inserm, Institut Necker enfants malades, Paris, France) du 3 au 8 juin 2019

Croissance des cellules beta pancréatiques, dont le rôle est d’intégrer les niveaux des nutriments et secréter l’insuline – Pancreatic beta cell growth, whose role is to integrate nutrient levels and secrete insulin.

Résumé  
La réunion a rassemblé vingt scientifiques internationaux de renom pour discuter les récents progrès sur notre compréhension de la façon dont les cellules détectent et réagissent aux nutriments. L’impact de ces mécanismes sur la croissance et le métabolisme a été particulièrement considéré.  Neuf scientifiques étaient européens et onze américains, dont trois jeunes chefs de groupe. Un éditeur de la revue Nature était également présent tout au long de la réunion. Les scientifiques ont couvert un large éventail de disciplines, notamment le métabolisme, la nutrition, l’évolution, la biologie structurale, la pharmacologie et la médecine. Ils ont discuté des connaissances fondamentales sur les mécanismes de détection des nutriments chez les eucaryotes et de leurs implications biomédicales pour le cancer, les maladies métaboliques et le vieillissement.

Summary

The meeting gathered twenty renowned international scientists to discuss advances in our understanding of how cells sense and respond to nutrients to impact cell metabolism and growth. Nine scientists were European and eleven American, including three young Group Leaders. One editor from the Nature journal was also present throughout the meeting. The scientists covered a broad spectrum of disciplines, including metabolism, nutrition, evolution, structural biology, pharmacology and medicine. The scientists discussed the fundamental knowledge of nutrient sensing mechanisms in eukaryotes and the biomedical implications for cancer, metabolic diseases and ageing.

 

Compte-rendu (en anglais) / Report

Michael Hall (Biozentrum, Switzerland) presented three separate studies on the roles of mTOR in cancer and diabetes. The first was onhistidine phosphorylation in tumorigenesis. Histidine phosphorylation is extremely labile and thus often called the “hidden phosphoproteome.” In particular, Hall described recent experiments with an mTOR-driven mouse model for hepatocellular carcinoma demonstrating that the protein histidine phosphatase LHPP is a novel tumor suppressor, suggesting that histidine phosphorylation is oncogenic.  Thesecondstudy addressed the effector pathways via which mTOR promotes cancer, showing that mTORC2 does so via activation of cardiolipin and glucosylceramide synthesis.  Thethirdstudy asked the question, how does obesity cause diabetes? Hall presented unpublished findings suggesting that an early step in the establishment of insulin insensitivity in response to a high fat diet is, surprisingly, not loss of mTOR signaling but rather loss of glycolysis.

David Sabatini (MIT, USA) presented work on the regulation of the mTOR Complex 1 (mTORC1) pathway by nutrients and the relationship of the nutrient-sensing pathway to lysosomes. He began by providing a brief overview of the organization of the pathway at the lysosomal surface and the key role the heterodimeric Rag GTPases in allowing mTORC1 to translocate to the lysosome in response to nutrient-rich conditions, where it can encounter its activator Rheb. He described the roles of several multi-component complexes, including GATOR1, GATOR2, Ragulator, and KICSTOR, in controlling the nucleotide loading state of the Rag GTPases. He then showed a structure solved by CryoEM of mTORC1 bound to Rags and Ragulator, which he proposed is what the complex looks like when bound to lysosomes. He then moved to the question of what is the nature of the nutrient sensors and went on to describe sensors for leucine (Sestrin2), arginine (CASTOR1) and S-adenosyl methionine (SAMTOR). He concluded his presentation with new work on the role and organization of the nutrient sensing pathway in vivo, with a focus on the liver. He provided evidence that nutrient sensing is compartmentalized and suggested that this may allow the pathway to regulate particular metabolic processes in only specialized cells. He also showed that both in the mouse liver and Drosophila fat body, Sestrin2 is required for mTORC1 to sense the absence of dietary leucine.

Brendan Manning (Harvard, USA) presented unpublished studies on the upstream regulation of mTORC1 by the tuberous sclerosis complex (TSC) protein complex and downstream functions of mTORC1 related to the control of lipid metabolism. He showed that multiple distinct signaling pathways that propagate signals from the cell surface to activate mTORC1, do so by controlling the subcellular localization of the TSC complex with Rheb at the lysosomal surface, where Rheb activates mTORC1, which is brought there independently in response to amino acids. He also showed new data in cultured hippocampal neurons that subpopulations of mTOR, Rheb, and the TSC complex colocalize with lysosomal marker in the soma, dendrites, and axons. Manning then turned his attention to the downstream metabolic effects of mTORC1 activation and inhibition. Building off his labs previous findings that mTORC1 induces de novo lipid synthesis via activation of the SREBP family of transcription factors, he presented lipidomic experiments demonstrating the effects of mTORC1 signaling on the lipid content of cells. His new findings suggest that mTORC1 controls the balance between membrane (phospholipids) and storage (triglycerides) species of lipids in cells and tumors, effects that alter fuel utilization and cell growth.

John Blenis (Cornell Medical, USA) focused his presentation on two stories centered on the metabolite, methylmalonic acid (MMA). First, he discussed the molecular basis of how metastasis inducers alter propionate metabolism to generate MMA during the transition of epithelial-like cancers to more aggressive mesenchymal cancers.  He then showed that MMA levels are significantly elevated in the serum of the elderly, and that MMA was required and sufficient to promote cancer progression. Dr. Blenis also showed that addition of MMA to a variety of epithelial cancer cells promotes the acquisition of an aggressive phenotype in cell culture and in mouse models suggesting that it may be a novel metabolic tumor progression and metastasis promoter.

Robbie Loewith (University of Geneva, Switzerland) spoke about the regulation of the two yeast TOR complexes.  In the first part of his talk he presented his group’s recent finding that glucose starvation triggers the polymerization of TORC1 particles, previously diffusely attached to the limiting membrane of the vacuole, into a huge helix they refer to as a TOROID (TORC1 Organized into Inhibited Domains). He explained how TORC1 becomes inactive when packaged into a TOROID and highlighted the fact that, by recruiting FKBP12, rapamycin inactivates TORC1 in a very similar fashion.  He next presented an unpublished high-resolution structure of the TOROID and outlined the near-future goal of the lab to observe the TOROID structure in situusing cryo-electron tomography.  The second part of his presentation concerned TORC2.  First, he described the structure of TORC2 and how from this structure his group learned to genetically reprogramme yeast cells such that TORC2 and not TORC1 is inhibited by rapamycin.  Next, he described a hit, palmitoylcarnitine, from a high throughput screening campaign for molecules that specifically inhibit TORC2. Characterization of the MOA of palmitoylcarnitine suggested that it inhibits TORC2 by integrating into the plasma membrane and reducing membrane tension.  Loss of tension causes a phase separation of PI (4,5) P2 which clusters and inactivates TORC2 in a manner analogous to TOROID formation.  Finally, he presented unpublished work describing the specificity of palmitoylcarnitine and the endogenous proteins required for its insertion into the membrane.

Roberto Zoncu (UC Berkley, USA) presented recent progress on two major directions in the lab. One is to elucidate the mechanisms via which mTORC1 senses cholesterol, an essential building block for membrane biogenesis and cell growth. Roberto discussed the role of physical contacts between lysosomes and the endoplasmic reticulum in delivering cholesterol from the ER to the lysosomal membrane, where the mTORC1-scaffolding machinery resides. Unique among many sterol carriers in the cell, oxysterol-binding protein (OSBP) is shown to be essential for the ability of mTORC1 to respond to cholesterol inputs via the Rag GTPases. Moreover, OSBP becomes aberrantly activated in the neurodegenerative and metabolic disease, Niemann-Pick type C (NPC), driving excess mTORC1 activation and autophagy impairment. Consequently, mTORC1 inhibition is shown to be sufficient to restore autophagic function in NPC cells. Zoncu then presented the application of chemical biology to dissect key regulatory steps in mTORC1 signaling. A screen with cysteine-reactive compounds yielded EN6, a small molecule that covalently modifies a conserved cysteine within the catalytic A subunit of the vacuolar H+ATPase (v-ATPase). Unlike other v-ATPase-targeting compounds, EN6 is shown to stimulate, rather than inhibit, proton-pumping and lysosomal acidification. Moreover, covalent v-ATPase modification by EN6 prevents mTORC1 from responding to nutrient inputs, causing its dissociation from the lysosome and the resulting activation of autophagy. Thus, EN6 emerges as a unique tool that can stimulate cellular clearance via both mTORC1-dependent and independent mechanisms. It also suggests that a conformational change powered by the ATP-hydrolyzing activity of the v-ATPase enables activation of the mTORC1-scaffolding machinery.

Reuben Shaw (Salk Institute, USA) discussed the AMPK signaling pathway, which is an ancient highly conserved energy sensor that reprograms cell growth and cell metabolism when nutrients and energy levels are low.  A decade of research has revealed two major outputs of the AMPK pathways when energy is low: 1) its suppression of mTORC1 signaling via direct phosphorylation of TSC2 and Raptor and 2) AMPK-dependent control of mitochondrial homeostasis via stimulation of autophagy and mitochondrial fission.  Upon prolonged nutrient stress, AMPK promotes gene expression changes that result in unregulated lysosome and mitochondrial biogenesis. Shaw presented his lab’s most recent unpublished studies focused on identifying the critical substrates of AMPK that mediate its effects on this transcriptional rewiring of metabolism.   

Dario Alessi (Dundee Uni. Scotland) presented his research on the LRRK2 enzyme is one of the most commonly mutated genes that causes familial inherited Parkinson’s. He discussed research that resulted in the identification of direct physiological substrates of LRRK2 that comprise a subgroup of around 14 Rab proteins. He demonstrated that all pathogenic LRRK2 mutations lead to enhanced Rab protein phosphorylation, consistent with Rab substrates bearing relevance to Parkinson’s disease. This phosphorylation alters the ability of Rab proteins to interact with cognate effectors such as RILPL1 and RILPL2 that interact with LRRK2 phosphorylated Rab proteins with greatly increased affinity. He showed data supporting the notion that Rab29 and VPS35 retromer complex act as major upstream regulators of LRRK2. In addition he presented work that a PPM1 family phosphatase PPM1H may act to dephosphorylate LRRK2 phosphorylated Rab proteins and that infection of macrophages with Candida Albicans induced LRRK2 to phosphorylate Rab10.

Kun-Liang Guan (UCSD, USA) started with an overview of the Hippo pathway in cellular regulation and tumorigenesis. He presented data of Hippo pathway regulation by cellular energy status (glucose) and the role of YAP, which is the key downstream effector of the Hippo pathway, in amino acid uptake to influence mTORC1. The second part of Guan’s presentation focused on the biochemical mechanism of Hippo pathway regulation. He showed that the STRIPAK complex integrates upstream signals to initiate the Hippo kinase cascade. He proposed that it is a protein phosphatase complex, being STRIPAK, that initiates the STE20 family protein kinase cascades, including the Hippo pathway.

Nathalie Spassky (ENS, France) presented work from her lab characterizing the mechanisms of multiciliated ependymal cells, which are lining the ventricular walls of the mammalian brain. These cells are derived from radial glial cells through symmetric terminal divisions or asymmetric divisions giving rise to adult neural stem cells. She presented evidence that the Geminin family members regulate the fate of these cells in vivo. The second half of the presentation was dedicated to the description of published and unpublished results using an in vitro assay of developing ependymal cells and the role of TORC1 in controlling the amplification of centrioles both in vivoand in vitro.

Navdeep S. Chandel (Northwestern University, USA) presented an unpublished story on how mitochondrial complex I controls neuronal function. Mitochondrial complex I is a 45-subunit complex that has two key functions. First, it transfers electrons from NADH to ubiquinone thereby regenerating NAD+ to allow the TCA cycle to function. Second, it contributes 40% to ATP generation by proton pumping. It is unknown the role of mitochondrial complex I linked NAD+ regeneration or ATP production for neuronal function. His findings indicate that mitochondrial complex I linked NAD+ regeneration in the brain is sufficient to prevent microglia activation, seizures and breathing abnormalities. However, NAD+ regeneration is NOT sufficient to prevent ataxia.  

Mario Pende (Inserm, Necker Institute, France) discussed the fruitful interaction of basic science and medical research in the field of nutrition, metabolism and growth. Monogenic diseases, such as Cloves syndromes and Tuberous Sclerosis Complex (TSC), had a tremendous care improvement due to the knowledge of signal transduction and development of specific kinase inhibitors. He presented unpublished data on cellular and molecular responses involved in two common manifestations of diseases with activated mTORC1: brain seizures and polycystic kidneys. His data showed that an mTORC1 target, S6 kinase 1, controls neuronal plasticity and the orientation of epithelial cell division.

Karen Vousden (Francis Crick Inst., UK) discussed the role of serine metabolism and the regulation of reactive oxygen species in cancer development and progression. She showed that limiting dietary serine and glycine lowers the circulating levels of these amino acids and retards tumour development in some preclinical models. Combining this approach with an inhibitor of the de novo serine synthesis pathway improved the therapeutic response. She also described a role for the mitochondrial enzyme ALDH1L2 in the regulation of formate overflow, providing evidence that formate released from tumour cells can promote invasion of both cancer and immune cells.

Sarah-Maria Fendt (VIB-KU Leuven, Belgium) presented her research on how metabolic changes regulate metastasis formation. She presented data showing that pyruvate is an important nutrient in the lung metastatic niche and that cancer cells require it to successfully undergo metastasis formation. In particular, pyruvate is important to enable extracellular matrix metabolism and to activate growth signaling. Moreover, she showed that serine and fatty acid metabolism are essential to support metastatic dissemination and seeding. In conclusion, her work provides evidence that targeting metabolism is an exciting strategy to prevent metastatic progression.

Heather Christofk (UCLA, USA) continued on the topic of cancer metabolism by discussing her lab’s approach of studying metabolic rewiring during viral infection as a way to identify critical metabolic enzymes for anabolism.  Using this approach, her lab found that asparagine synthetase (ASNS) upregulation is required for optimal adenovirus replication, and that one of the ASNS products, asparagine, is limiting for cancer cell mTORC1 activation, anabolism, and proliferation.  To target cancer asparagine dependence, her lab coupled use of L-Asparaginase or an asparagine-low diet to reduce circulating asparagine levels with complex I inhibitors (metformin or IACS-010759) to limit cellular aspartate production, an obligate substrate for ASNS production of asparagine.  These combinations were sufficient to reduce tumor asparagine levels, mTORC1 activity, and growth in multiple cancer xenograft models.  Furthermore, data were presented that aspartate-derived asparagine may communicate respiration to mTORC1 in cells. 

Pierre Léopold (Institut Curie, Paris, France) presented recent work on the integration of the nutrient response at the organismal level. His work in Drosophila highlights the role played by relay organs, like the fat body (liver and fat homolog for insects) and the brain in orchestrating the response to amino acids for growth and tissue homeostasis. He reported the identification of several fat body-derived factors that control the release of insulins from brain neurosecretory cells. He also presented his latest work on the role of adiponectin receptor signaling in the brain linking high energy diet to peripheral insulin resistance.

Linda Partridge (UCL, UK/Max Planck Cologne, Germany) spoke about the role of nutrient-sensing pathways in healthy ageing. Dietary, genetic and pharmacological interventions can promote heathy ageing and combat age-related diseases in laboratory animals. Among the most powerful of these are the drug sirolimus and dietary restriction (DR), both of which can give health benefits in older people. For these interventions, or interventions derived from them, to be maximally effective in humans, it is important to determine the optimal dosing regime. Both sirolimus and DR can have undesirable side effects, and by minimizing the dose and the length of time for which it is applied, it may be possible to maximize the ratio of benefits to side-effects. Experiments with both the fruit fly Drosophila and with mice have indicated that there is an extraordinarily long-term effect of earlier treatment with the drug, with the structure and function of the gut showing a major improvement. The mechanism appears to be an almost permanent elevation in autophagy, a mechanism of cellular clearance, which may maintain the cells of the gut in a youthful state. In mice, intermittent dosing with sirolimus abolished its adverse, metabolic side-effects. Highly intermittent dosing with sirolimus thus appears to be capable of achieving its full benefits, with minimal or no adverse effects. In contrast, evidence from mice suggests that for DR to be maximally effective it should be applied from mid-adulthood, since after that the mice become unresponsive, showing no increase in lifespan. This refractoriness is associated with failure of gene expression patterns in the white adipose tissue (fat) to respond to the intervention. For both interventions we need to understand more about the molecular basis of the persisting effects of earlier experience.

Anne Brunet (Stanford, USA) introduced the adult brain, which comprises neural stem cell (NSC) pools with quiescent and activated populations. During aging, the ability of NSC to transition from quiescence to activation drastically decreased. Her transcriptomic analysis revealed that quiescent and activated NSCs exhibited differences in their protein homeostasis network. Whereas activated NSCs had active proteasomes, quiescent NSCs contained large lysosomes. Quiescent NSCs from young mice accumulated protein aggregates, and many of these aggregates were stored in large lysosomes. Perturbation of lysosomal activity in quiescent NSCs affected protein-aggregate accumulation and the ability of quiescent NSCs to activate. During aging, quiescent NSCs displayed defects in their lysosomes, increased accumulation of protein aggregates, and reduced ability to activate. Enhancement of the lysosome pathway in old quiescent NSCs cleared protein aggregates and ameliorated the ability of quiescent NSCs to activate, allowing them to regain a more youthful state. Brunet’s lab is currently characterizing the protein aggregates in young and old NSCs, with the goal of rejuvenating the old neurogenic niche and counteract decline in brain function with aging. 

Mustafa Sahin (Boston Children’s Hospital/Harvard, USA) discussed the role of the mTOR pathway in autism spectrum disorder, focusing on Tuberous Sclerosis Complex. He presented three stories on the role of TSC1/TSC2 protein in neuronal development and function. First, loss of TSC1/TSC2 affects axonal specification and targeting in both cell culture and in mouse brain. Second, he shared unpublished data on a stem-cell derive cortical neuron model, demonstrating that loss of TSC2 is associated with hyperexcitability. Finally, he described a Purkinje-cell specific knockout of Tsc1 gene resulting in autistic-like behavior in mice. This experimental model was instrumental in defining critical periods for treatment with mTOR inhibitors to rescue the behavioral deficits, and thus provided insights about design of clinical trials in patients affects with TSC and autism.

Dr. Helen Bateup (UC Berkeley, USA) presented her work on mouse and human stem cell-derived models of Tuberous Sclerosis Complex (TSC). She showed that deletion of the Tsc1gene and upregulation of mTORC1 signaling in dopamine neurons in mice results in profound somatodendritic and axonal hypertrophy. These structural changes severely impair dopamine release in the striatum, which is sufficient to impair cognitive flexibility in a reversal learning task. In addition, Dr. Bateup discussed the development of human stem cell-derived brain organoid models of TSC. She showed that complete loss of theTSC1 or TSC2genes early in neural development leads to the formation of dysplastic cells that resemble cortical tuber cells found in TSC patients. Additionally, bidirectional changes in mTORC1 signaling strongly impact the balance of neuron and astrocyte differentiation in human brain organoids. Together, Dr. Bateup’s presentation revealed the multifaceted roles of Tsc1-mTORC1 signaling on neuronal development, structure, and function and provided insights to potential disease mechanisms in mTOR-related disorders.

 

Communications

  • Dario Alessi (Dundee University, UK) : LRRK2 and Rab Biology of Parkinson’s
  • Hélène Bateup (Berkeley University, USA) : The multi-faceted roles of TSC-mTOR signaling in neuronal development, function and disease
  • John Blenis (Weill Cornell Medicine, New York, USA) : mTORC1 and S6K1: metabolism, RNA processing and cell growth control
  • Anne Brunet (Dept of Genetics, Stanford University, USA): Metabolic regulation of aging stem cells
  • Navdeep Chandel (Northwestern University, Chicago, USA): Mitochondria as signaling organelles
  • HeatherChristofk (University of Los Angeles, USA) : Metabolic transitions in cancer
  • Sarah-MariaFendt (VIB – KU Leuven Center for Cancer biology, Belgium) : The role of metabolism in metastasis formation
  • Kun-LiangGuan (University of California San Diego, USA) : The Hippo pathway
  • Michael Hall (N. Biozentrum, University of Basel, Switzerland) : mTOR signaling in growth and metabolism
  • Pierre Leopold (INSERM, CNRS, Paris, France) : Nutrient sensing and development
  • Robbie Loewith (University of Geneva, Switzerland) : Drugging membranes to inhibit TOR
  • Brendan D.Manning (Harvard University, Boston, USA) : Growth signal integration and metabolic regulation
  • Linda Partridge (UCL Cologna, Germany) : Nutrient sensing: a gut feeling
  • Mario Pende (Inserm, Institut Necker Enfants Malades, Paris, France) : Growth control by S6K1 and YAP
  • David M. Sabatini (Whitehead Institute for Biomedical Research, Cambridge, USA) : mTOR and Lysosomes in Growth Control
  • Mustafa Sahin (Boston Children’s hospital Boston, USA) : Cell autonomous and non autonomous roles of TSC1/2 protein complex in neurons
  • Reuben Shaw (Salk Institute for Biological Studies, La Jolla, USA) : AMPK: good signals in bad times
  • Nathalie Spassky (INSERM, Paris) : Common origins of adult neural stem cells and multiciliated ependymal cells
  • Karen Vousden (Francis Crick Institute, London, UK) : A role for ROS in tumour progression and metastasis
  • Roberto Zoncu: (Berkeley University, USA) : Novel roles for the lysosome in nutrient sensing and signal transduction
  • Invited journalist: Marte Barbara (Editor), Nature Magazine, London, UK