Retinal ganglion cells (RGCs) are the sole projecting neurons of the retina and their axons form the optic nerve. Here, we show that embryogenesis-associated mouse RGC differentiation depends on mitophagy, the programmed autophagic clearance of mitochondria. The elimination of mitochondria during RGC differentiation was coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes at the mRNA level. Pharmacological and genetic inhibition of either mitophagy or glycolysis consistently inhibited RGC differentiation. Local hypoxia triggered expression of the mitophagy regulator BCL2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L, best known as NIX) at peak RGC differentiation. Retinas from NIX-deficient mice displayed increased mitochondrial mass, reduced expression of glycolytic enzymes and decreased neuronal differentiation. Similarly, we provide evidence that NIX-dependent mitophagy contributes to mitochondrial elimination during macrophage polarization towards the proinflammatory and more glycolytic M1 phenotype, but not to M2 macrophage differentiation, which primarily relies on oxidative phosphorylation. In summary, developmentally controlled mitophagy promotes a metabolic switch towards glycolysis, which in turn contributes to cellular differentiation in several distinct developmental contexts.

Blocking mitotic progression has been proposed as an attractive therapeutic strategy to impair proliferation of tumour cells. However, how cells survive during prolonged mitotic arrest is not well understood. We show here that survival during mitotic arrest is affected by the special energetic requirements of mitotic cells. Prolonged mitotic arrest results in mitophagy-dependent loss of mitochondria, accompanied by reduced ATP levels and the activation of AMPK. Oxidative respiration is replaced by glycolysis owing to AMPK-dependent phosphorylation of PFKFB3 and increased production of this protein as a consequence of mitotic-specific translational activation of its mRNA. Induction of autophagy or inhibition of AMPK or PFKFB3 results in enhanced cell death in mitosis and improves the anti-tumoral efficiency of microtubule poisons in breast cancer cells. Thus, survival of mitotic-arrested cells is limited by their metabolic requirements, a feature with potential implications in cancer therapies aimed to impair mitosis or metabolism in tumour cells.

Glioblastoma (GB) is the most lethal brain tumor, and Wingless (Wg)-related integration site (WNT) pathway activation in these tumors is associated with a poor prognosis. Clinically, the disease is characterized by progressive neurological deficits. However, whether these symptoms result from direct or indirect damage to neurons is still unresolved. Using Drosophila and primary xenografts as models of human GB, we describe, here, a mechanism that leads to activation of WNT signaling (Wg in Drosophila) in tumor cells. GB cells display a network of tumor microtubes (TMs) that enwrap neurons, accumulate Wg receptor Frizzled1 (Fz1), and, thereby, deplete Wg from neurons, causing neurodegeneration. We have defined this process as “vampirization.” Furthermore, GB cells establish a positive feedback loop to promote their expansion, in which the Wg pathway activates cJun N-terminal kinase (JNK) in GB cells, and, in turn, JNK signaling leads to the post-transcriptional up-regulation and accumulation of matrix metalloproteinases (MMPs), which facilitate TMs’ infiltration throughout the brain, TMs’ network expansion, and further Wg depletion from neurons. Consequently, GB cells proliferate because of the activation of the Wg signaling target, β-catenin, and neurons degenerate because of Wg signaling extinction. Our findings reveal a molecular mechanism for TM production, infiltration, and maintenance that can explain both neuron-dependent tumor progression and also the neural decay associated with GB.

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Autophagic and endo-lysosomal degradative pathways are essential for cell homeostasis. Availability of reliable tools to interrogate these pathways is critical to unveil their involvement in physiology and pathophysiology. Although several probes have been recently developed to monitor autophagic or lysosomal compartments, their specificity has not been validated through co-localization studies with well-known markers. Here, we evaluate the selectivity and interactions between one lysosomal (Lyso-ID) and one autophagosomal (Cyto-ID) probe under conditions modulating autophagy and/or endo-lysosomal function in live cells. The probe for acidic compartments Lyso-ID was fully localized inside vesicles positive for markers of late endosome-lysosomes, including Lamp1-GFP and GFP-CINCCKVL. Induction of autophagy by amino acid deprivation in bovine aortic endothelial cells caused an early and potent increase in the fluorescence of the proposed autophagy dye Cyto-ID. Cyto-ID-positive compartments extensively co-localized with the autophagosomal fluorescent reporter RFP-LC3, although the time and/or threshold for organelle detection was different for each probe. Interestingly, use of Cyto-ID in combination with Lysotracker Red or Lyso-ID allowed the observation of structures labeled with either one or both probes, the extent of co-localization increasing upon treatment with protease inhibitors. Inhibition of the endo-lysosomal pathway with chloroquine or U18666A resulted in the formation of large Cyto-ID and Lyso-ID-positive compartments. These results constitute the first assessment of the selectivity of Cyto-ID and Lyso-ID as probes for the autophagic and lysosomal pathways, respectively. Our observations show that these probes can be used in combination with protein-based markers for monitoring the interactions of both pathways in live cells.

RINGO/Speedy proteins are direct activators of Cdk1 and Cdk2 that have no sequence homology to cyclins. We have characterized the role in cell-cycle progression of a new human member of this protein family referred to as RINGO C. We show that siRNA-mediated knockdown of RINGO C results in premature mitotic exit with misaligned chromosomes, even in the presence of microtubule poisons. Time-lapse-microscopy experiments suggest that RINGO C is involved in the spindle-assembly checkpoint (SAC). Consistent with this idea, RINGO-C-depleted cells show impaired recruitment of the SAC components Mad2, Bub1 and BubR1. As the checkpoint is overridden, cells display defective chromosome segregation, which leads to an increased number of micronuclei and binucleated structures. Intriguingly, we found that RINGO C can associate with the mitotic kinase Aurora B, and downregulation of RINGO C produces mislocalization of the active form of Aurora B in prometaphase. Taken together, our results indicate a role for RINGO C in the mitotic checkpoint, which might be mediated by defective recruitment of SAC components and deregulation of the activity of Aurora kinase B.

22Glioblastoma (GB) is the most lethal brain tumor due to its high proliferation, 23 aggressiveness, infiltration capacity and resilience to current treatments. Activation of 24 the Wingless-related-integration-site (WNT) pathway is associated with a bad 25 prognosis. Using Drosophila and primary xenograft models of human GB, we describe 26 a mechanism that leads to the activation of WNT signaling [Wingless (Wg) in 27 Drosophila] in tumor cells. GB cells display a network of tumor microtubes (TMs) which 28 enwraps neurons, accumulates Wg receptor Frizzled1 (Fz1), and, thereby, actively 29 depletes Wg from the neurons. Consequently, GB cells proliferate due to β-catenin 30 activation, and neurons degenerate due to Wg signaling extinction. This novel view 31 explains both neuron-dependent tumor progression, and also the neural decay 32 associated with GB. 33 Keywords 34Neuron, Glia, Cancer, wingless, Frizzled, Glioblastoma, 35 Cytoneme, Wg-depletion. 36 37The WNT canonical pathway is activated upon the ligand "Wingless-related integration 48 site" (WNT) binding to specific receptors (LRPs and FZD) in the plasma membrane. As 49 a consequence, the destruction complex (APC and Axin) is inactivated and β-catenin 50 (armadillo in Drosophila) is released. Further, β-catenin translocates into the cell 51 nucleus where it promotes the expression of target genes (i.e. Cyclin D1 and Myc) 7-8 . 52The WNT pathway is conserved through metazoans and it plays a central role in brain 53 development 9 , adult neuronal physiology 10 and synaptogenesis 11 . Perturbations in 54WNT signaling are associated with neural deficits, Alzheimer´s disease and brain 55 cancer, most notably GB 12 . WNT and FZD signaling can be deregulated in 56 glioblastoma 13-14 (reviewed in 15 ). In particular, one of the hallmarks of bad prognosis is 57 the accumulation of ß-catenin in tumoral cells [16][17] , indicating an activation of WNT/FZD 58 pathway 18 . 59 GB cells extend ultra-long membrane protrusions that interconnect tumor cells 19 . 60These tumor microtubes (TMs) are associated with the worst prognosis in molecular 61 subtypes of human gliomas. TMs contribute to invasion and proliferation, resulting in 62 effective brain colonization by GB cells. Moreover, TMs constitute a multicellular 63 network that connects GB cells over long distances, a feature that likely provides 64 resistance against radiotherapy, chemotherapy and surgery [19][20] . Considering the many 65 cytological similarities of TMs and tunneling nanotubes (TNTs) 21 , it seems that TMs in 66 aggressive gliomas are the in vivo correlate of TNTs described in cell culture. In 67 addition, TMs seem akin to a basic mechanism of cell-cell connection and molecular 68 communication called "cytonemes" in Drosophila 22 . Growth Associated Protein-43 69 (GAP43) is essential for the development of TMs and, thus, the tumor cell network 70 which is associated with GB progression 19 . However, many aspects of this 71 paradigmatic finding in glioma biology are still unexplored, including its i...

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