Malonyl-CoA is a conserved endogenous ATP-competitive mTORC1 inhibitor.

Cell growth is regulated by the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which functions both as a nutrient sensor and a master controller of virtually all biosynthetic pathways. This ensures that cells are metabolically active only when conditions are optimal for growth. Notably, although mTORC1 is known to regulate fatty acid biosynthesis, how and whether the cellular lipid biosynthetic capacity signals back to fine-tune mTORC1 activity remains poorly understood. Here we show that mTORC1 senses the capacity of a cell to synthesise fatty acids by detecting the levels of malonyl-CoA, an intermediate of this biosynthetic pathway. We find that, in both yeast and mammalian cells, this regulation is direct, with malonyl-CoA binding to the mTOR catalytic pocket and acting as a specific ATP-competitive inhibitor. When fatty acid synthase (FASN) is downregulated/inhibited, elevated malonyl-CoA levels are channelled to proximal mTOR molecules that form direct protein-protein interactions with acetyl-CoA carboxylase 1 (ACC1) and FASN. Our findings represent a conserved and unique homeostatic mechanism whereby impaired fatty acid biogenesis leads to reduced mTORC1 activity to coordinately link this metabolic pathway to the overall cellular biosynthetic output. Moreover, they reveal the existence of a physiological metabolite that directly inhibits the activity of a signalling kinase in mammalian cells by competing with ATP for binding.

Interferon regulates neural stem cell function at all ages by orchestrating mTOR and cell cycle.

Stem cells show intrinsic interferon signalling, which protects them from viral infections at all ages. In the ageing brain, interferon signalling also reduces the ability of stem cells to activate. Whether these functions are linked and at what time interferons start taking on a role in stem cell functioning is unknown. Additionally, the molecular link between interferons and activation in neural stem cells and how this relates to progenitor production is not well understood. Here we combine single-cell transcriptomics, RiboSeq and mathematical models of interferon to show that this pathway is important for proper stem cell function at all ages in mice. Interferon orchestrates cell cycle and mTOR activity to post-transcriptionally repress Sox2 and induces quiescence. The interferon response then decreases in the subsequent maturation states. Mathematical simulations indicate that this regulation is beneficial for the young and harmful for the old brain. Our study establishes molecular mechanisms of interferon in stem cells and interferons as genuine regulators of stem cell homeostasis and a potential therapeutic target to repair the ageing brain.

Brain-enriched RagB isoforms regulate the dynamics of mTORC1 activity through GATOR1 inhibition.

Mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient availability to appropriately regulate cellular anabolism and catabolism. During nutrient restriction, different organs in an animal do not respond equally, with vital organs being relatively spared. This raises the possibility that mTORC1 is differentially regulated in different cell types, yet little is known about this mechanistically. The Rag GTPases, RagA or RagB bound to RagC or RagD, tether mTORC1 in a nutrient-dependent manner to lysosomes where mTORC1 becomes activated. Although the RagA and B paralogues were assumed to be functionally equivalent, we find here that the RagB isoforms, which are highly expressed in neurons, impart mTORC1 with resistance to nutrient starvation by inhibiting the RagA/B GTPase-activating protein GATOR1. We further show that high expression of RagB isoforms is observed in some tumours, revealing an alternative strategy by which cancer cells can retain elevated mTORC1 upon low nutrient availability.

G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling.

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.

Metabolites Regulate Cell Signaling and Growth via Covalent Modification of Proteins.

Metabolites affect cell growth in two different ways. First, they serve as building blocks for biomass accumulation. Second, metabolites regulate the activity of growth-relevant signaling pathways. They do so in part by covalently attaching to proteins, thereby generating post-translational modifications (PTMs) that affect protein function, the focus of this Perspective. Recent advances in mass spectrometry have revealed a wide variety of such metabolites, including lipids, amino acids, Coenzyme-A, acetate, malonate, and lactate to name a few. An active area of research is to understand which modifications affect protein function and how they do so. In many cases, the cellular levels of these metabolites affect the stoichiometry of the corresponding PTMs, providing a direct link between cell metabolism and the control of cell signaling, transcription, and cell growth.

Cdk4 and Cdk6 Couple the Cell-Cycle Machinery to Cell Growth via mTORC1.

Cell growth is coupled to cell-cycle progression in mitotically proliferating mammalian cells, but the underlying molecular mechanisms are not well understood. CyclinD-Cdk4/6 is known to phosphorylate RB to promote S-phase entry, but recent work suggests they have additional functions. We show here that CyclinD-Cdk4/6 activates mTORC1 by binding and phosphorylating TSC2 on Ser1217 and Ser1452. Pharmacological inhibition of Cdk4/6 leads to a rapid, TSC2-dependent reduction of mTORC1 activity in multiple human and mouse cell lines, including breast cancer cells. By simultaneously driving mTORC1 and E2F, CyclinD-Cdk4/6 couples cell growth to cell-cycle progression. Consistent with this, we see that mTORC1 activity is cell cycle dependent in proliferating neural stem cells of the adult rodent brain. We find that Cdk4/6 inhibition reduces cell proliferation partly via TSC2 and mTORC1. This is of clinical relevance, because Cdk4/6 inhibitors are used for breast cancer therapy.

Ral GTPases in Schwann cells promote radial axonal sorting in the peripheral nervous system.

Small GTPases of the Rho and Ras families are important regulators of Schwann cell biology. The Ras-like GTPases RalA and RalB act downstream of Ras in malignant peripheral nerve sheath tumors. However, the physiological role of Ral proteins in Schwann cell development is unknown. Using transgenic mice with ablation of one or both Ral genes, we report that Ral GTPases are crucial for axonal radial sorting. While lack of only one Ral GTPase was dispensable for early peripheral nerve development, ablation of both RalA and RalB resulted in persistent radial sorting defects, associated with hallmarks of deficits in Schwann cell process formation and maintenance. In agreement, ex vivo-cultured Ral-deficient Schwann cells were impaired in process extension and the formation of lamellipodia. Our data indicate further that RalA contributes to Schwann cell process extensions through the exocyst complex, a known effector of Ral GTPases, consistent with an exocyst-mediated function of Ral GTPases in Schwann cells.

Schwann cells, but not Oligodendrocytes, Depend Strictly on Dynamin 2 Function.

Myelination requires extensive plasma membrane rearrangements, implying that molecules controlling membrane dynamics play prominent roles. The large GTPase dynamin 2 (DNM2) is a well-known regulator of membrane remodeling, membrane fission, and vesicular trafficking. Here, we genetically ablated Dnm2 in Schwann cells (SCs) and in oligodendrocytes of mice. Dnm2 deletion in developing SCs resulted in severely impaired axonal sorting and myelination onset. Induced Dnm2 deletion in adult SCs caused a rapidly-developing peripheral neuropathy with abundant demyelination. In both experimental settings, mutant SCs underwent prominent cell death, at least partially due to cytokinesis failure. Strikingly, when Dnm2 was deleted in adult SCs, non-recombined SCs still expressing DNM2 were able to remyelinate fast and efficiently, accompanied by neuropathy remission. These findings reveal a remarkable self-healing capability of peripheral nerves that are affected by SC loss. In the central nervous system, however, we found no major defects upon Dnm2 deletion in oligodendrocytes.

mTORC1 Is Transiently Reactivated in Injured Nerves to Promote c-Jun Elevation and Schwann Cell Dedifferentiation.

Schwann cells (SCs) are endowed with a remarkable plasticity. When peripheral nerves are injured, SCs dedifferentiate and acquire new functions to coordinate nerve repair as so-called repair SCs. Subsequently, SCs redifferentiate to remyelinate regenerated axons. Given the similarities between SC dedifferentiation/redifferentiation in injured nerves and in demyelinating neuropathies, elucidating the signals involved in SC plasticity after nerve injury has potentially wider implications. c-Jun has emerged as a key transcription factor regulating SC dedifferentiation and the acquisition of repair SC features. However, the upstream pathways that control c-Jun activity after nerve injury are largely unknown. We report that the mTORC1 pathway is transiently but robustly reactivated in dedifferentiating SCs. By inducible genetic deletion of the functionally crucial mTORC1-subunit Raptor in mouse SCs (including male and female animals), we found that mTORC1 reactivation is necessary for proper myelin clearance, SC dedifferentiation, and consequently remyelination, without major alterations in the inflammatory response. In the absence of mTORC1 signaling, c-Jun failed to be upregulated correctly. Accordingly, a c-Jun binding motif was found to be enriched in promoters of genes with reduced expression in injured mutants. Furthermore, using cultured SCs, we found that mTORC1 is involved in c-Jun regulation by promoting its translation, possibly via the eIF4F-subunit eIF4A. These results provide evidence that proper c-Jun elevation after nerve injury involves also mTORC1-dependent post-transcriptional regulation to ensure timely dedifferentiation of SCs.SIGNIFICANCE STATEMENT A crucial evolutionary acquisition of vertebrates is the envelopment of axons in myelin sheaths produced by oligodendrocytes in the CNS and Schwann cells (SCs) in the PNS. When myelin is damaged, conduction of action potentials along axons slows down or is blocked, leading to debilitating diseases. Unlike oligodendrocytes, SCs have a high regenerative potential, granted by their remarkable plasticity. Thus, understanding the mechanisms underlying SC plasticity may uncover new therapeutic targets in nerve regeneration and demyelinating diseases. Our work reveals that reactivation of the mTORC1 pathway in SCs is essential for efficient SC dedifferentiation after nerve injury. Accordingly, modulating this signaling pathway might be of therapeutic relevance in peripheral nerve injury and other diseases.

De novo fatty acid synthesis by Schwann cells is essential for peripheral nervous system myelination.

Myelination calls for a remarkable surge in cell metabolism to facilitate lipid and membrane production. Endogenous fatty acid (FA) synthesis represents a potentially critical process in myelinating glia. Using genetically modified mice, we show that Schwann cell (SC) intrinsic activity of the enzyme essential for de novo FA synthesis, fatty acid synthase (FASN), is crucial for precise lipid composition of peripheral nerves and fundamental for the correct onset of myelination and proper myelin growth. Upon FASN depletion in SCs, epineurial adipocytes undergo lipolysis, suggestive of a compensatory role. Mechanistically, we found that a lack of FASN in SCs leads to an impairment of the peroxisome proliferator-activated receptor (PPAR) γ-regulated transcriptional program. In agreement, defects in myelination of FASN-deficient SCs could be ameliorated by treatment with the PPARγ agonist rosiglitazone ex vivo and in vivo. Our results reveal that FASN-driven de novo FA synthesis in SCs is mandatory for myelination and identify lipogenic activation of the PPARγ transcriptional network as a putative downstream functional mediator.

Myelination and mTOR.

Myelinating cells surround axons to accelerate the propagation of action potentials, to support axonal health, and to refine neural circuits. Myelination is metabolically demanding and, consistent with this notion, mTORC1-a signaling hub coordinating cell metabolism-has been implicated as a key signal for myelination. Here, we will discuss metabolic aspects of myelination, illustrate the main metabolic processes regulated by mTORC1, and review advances on the role of mTORC1 in myelination of the central nervous system and the peripheral nervous system. Recent progress has revealed a complex role of mTORC1 in myelinating cells that includes, besides positive regulation of myelin growth, additional critical functions in the stages preceding active myelination. Based on the available evidence, we will also highlight potential nonoverlapping roles between mTORC1 and its known main upstream pathways PI3K-Akt, Mek-Erk1/2, and AMPK in myelinating cells. Finally, we will discuss signals that are already known or hypothesized to be responsible for the regulation of mTORC1 activity in myelinating cells.

Dual function of the PI3K-Akt-mTORC1 axis in myelination of the peripheral nervous system.

Myelination is a biosynthetically demanding process in which mTORC1, the gatekeeper of anabolism, occupies a privileged regulatory position. We have shown previously that loss of mTORC1 function in Schwann cells (SCs) hampers myelination. Here, we genetically disrupted key inhibitory components upstream of mTORC1, TSC1 or PTEN, in mouse SC development, adult homeostasis, and nerve injury. Surprisingly, the resulting mTORC1 hyperactivity led to markedly delayed onset of both developmental myelination and remyelination after injury. However, if mTORC1 was hyperactivated after myelination onset, radial hypermyelination was observed. At early developmental stages, physiologically high PI3K-Akt-mTORC1 signaling suppresses expression of Krox20 (Egr2), the master regulator of PNS myelination. This effect is mediated by S6K and contributes to control mechanisms that keep SCs in a not-fully differentiated state to ensure proper timing of myelination initiation. An ensuing decline in mTORC1 activity is crucial to allow myelination to start, while remaining mTORC1 activity drives myelin growth.

mTORC1 controls PNS myelination along the mTORC1-RXRγ-SREBP-lipid biosynthesis axis in Schwann cells.

Myelin formation during peripheral nervous system (PNS) development, and reformation after injury and in disease, requires multiple intrinsic and extrinsic signals. Akt/mTOR signaling has emerged as a major player involved, but the molecular mechanisms and downstream effectors are virtually unknown. Here, we have used Schwann-cell-specific conditional gene ablation of raptor and rictor, which encode essential components of the mTOR complexes 1 (mTORC1) and 2 (mTORC2), respectively, to demonstrate that mTORC1 controls PNS myelination during development. In this process, mTORC1 regulates lipid biosynthesis via sterol regulatory element-binding proteins (SREBPs). This course of action is mediated by the nuclear receptor RXRγ, which transcriptionally regulates SREBP1c downstream of mTORC1. Absence of mTORC1 causes delayed myelination initiation as well as hypomyelination, together with abnormal lipid composition and decreased nerve conduction velocity. Thus, we have identified the mTORC1-RXRγ-SREBP axis controlling lipid biosynthesis as a major contributor to proper peripheral nerve function.

Synthesis and preliminary evaluation in tumor bearing mice of new (18)F-labeled arylsulfone matrix metalloproteinase inhibitors as tracers for positron emission tomography.

New fluorinated, arylsulfone-based matrix metalloproteinase (MMP) inhibitors containing carboxylate as the zinc binding group were synthesized as radiotracers for positron emission tomography. Inhibitors were characterized by Ki for MMP-2 in the nanomolar range and by a fair selectivity for MMP-2/9/12/13 over MMP-1/3/14. Two of these compounds were obtained in the (18)F-radiolabeled form, with radiochemical purity and yield suitable for preliminary studies in mice xenografted with a human U-87 MG glioblastoma. Target density in xenografts was assessed by Western blot, yielding Bmax/Kd = 14. The biodistribution of the tracer was dominated by liver uptake and hepatobiliary clearance. Tumor uptake of (18)F-labeled MMP inhibitors was about 30% that of [(18)F]fluorodeoxyglucose. Accumulation of radioactivity within the tumor periphery colocalized with MMP-2 activity (evaluated by in situ zimography). However, specific tumor uptake accounted for only 18% of total uptake. The aspecific uptake was ascribed to the high binding affinity between the radiotracer and serum albumin.

Practice of yoga may cause damage of both sciatic nerves: a case report.

Sciatic nerve traumatic damage very rarely occurs bilaterally. We describe the case of a 67-year-old woman who reported a bilateral traumatic lesion of the sciatic nerve during practice of yoga. Nerve conduction studies showed a bilateral sciatic nerve neuropathy, mostly affecting the peroneal component. Lumbar plexus MRI documented regular anatomical features of the main principal nerve roots with bilateral T2 signal alteration of roots L4, L5 and S1 that extended into the sciatic nerves showing both increase in size, probably related to chronic injury of nerves, and an alteration in diffusion signal that suggested a recent acute overlapped process.

MMP2-9 cleavage of dystroglycan alters the size and molecular composition of Schwann cell domains.

Myelinating glial cells exhibit a spectacular cytoarchitecture, because they polarize on multiple axes and domains. How this occurs is essentially unknown. The dystroglycan-dystrophin complex is required for the function of myelin-forming Schwann cells. Similar to other tissues, the dystroglycan complex in Schwann cells localizes with different dystrophin family members in specific domains, thus promoting polarization. We show here that cleavage of dystroglycan by matrix metalloproteinases 2 and 9, an event that is considered pathological in most tissues, is finely and dynamically regulated in normal nerves and modulates dystroglycan complex composition and the size of Schwann cell compartments. In contrast, in nerves of Dy(2j/2j) mice, a model of laminin 211 deficiency, metalloproteinases 2 and 9 are increased, causing excessive dystroglycan cleavage and abnormal compartments. Pharmacological inhibition of cleavage rescues the cytoplasmic defects of Dy(2j/2j) Schwann cells. Thus, regulated cleavage may be a general mechanism to regulate protein complex composition in physiological conditions, whereas unregulated processing is pathogenic and a target for treatment in disease.

Non-redundant function of dystroglycan and ß1 integrins in radial sorting of axons.

Radial sorting allows the segregation of axons by a single Schwann cell (SC) and is a prerequisite for myelination during peripheral nerve development. Radial sorting is impaired in models of human diseases, congenital muscular dystrophy (MDC) 1A, MDC1D and Fukuyama, owing to loss-of-function mutations in the genes coding for laminin α2, Large or fukutin glycosyltransferases, respectively. It is not clear which receptor(s) are activated by laminin 211, or glycosylated by Large and fukutin during sorting. Candidates are αß1 integrins, because their absence phenocopies laminin and glycosyltransferase deficiency, but the topography of the phenotypes is different and ß1 integrins are not substrates for Large and fukutin. By contrast, deletion of the Large and fukutin substrate dystroglycan does not result in radial sorting defects. Here, we show that absence of dystroglycan in a specific genetic background causes sorting defects with topography identical to that of laminin 211 mutants, and recapitulating the MDC1A, MDC1D and Fukuyama phenotypes. By epistasis studies in mice lacking one or both receptors in SCs, we show that only absence of ß1 integrins impairs proliferation and survival, and arrests radial sorting at early stages, that ß1 integrins and dystroglycan activate different pathways, and that the absence of both molecules is synergistic. Thus, the function of dystroglycan and ß1 integrins is not redundant, but is sequential. These data identify dystroglycan as a functional laminin 211 receptor during axonal sorting and the key substrate relevant to the pathogenesis of glycosyltransferase congenital muscular dystrophies.