Immunodetection of Truncated Forms of the α6 Subunit of the nAChR in the Brain of Spinosad Resistant Ceratitis capitata Phenotypes.

The α6 subunit of the nicotinic acetylcholine receptor (nAChR) has been proposed as the target for spinosad in insects. Point mutations that result in premature stop codons in the α6 gene of Ceratitis capitata flies have been previously associated with spinosad resistance, but it is unknown if these transcripts are translated and if so, what is the location of the putative truncated proteins. In this work, we produced a specific antibody against C. capitata α6 (Ccα6) and validated it by ELISA, Western blotting and immunofluorescence assays in brain tissues. The antibody detects both wild-type and truncated forms of Ccα6 in vivo, and the protein is located in the cell membrane of the brain of wild-type spinosad sensitive flies. On the contrary, the shortened transcripts present in resistant flies generate putative truncated proteins that, for the most part, fail to reach their final destination in the membrane of the cells and remain in the cytoplasm. The differences observed in the locations of wild-type and truncated α6 proteins are proposed to determine the susceptibility or resistance to spinosad.

Synaptic components are required for glioblastoma progression in Drosophila.

Glioblastoma (GB) is the most aggressive, lethal and frequent primary brain tumor. It originates from glial cells and is characterized by rapid expansion through infiltration. GB cells interact with the microenvironment and healthy surrounding tissues, mostly neurons and vessels. GB cells project tumor microtubes (TMs) contact with neurons, and exchange signaling molecules related to Wingless/WNT, JNK, Insulin or Neuroligin-3 pathways. This cell to cell communication promotes GB expansion and neurodegeneration. Moreover, healthy neurons form glutamatergic functional synapses with GB cells which facilitate GB expansion and premature death in mouse GB xerograph models. Targeting signaling and synaptic components of GB progression may become a suitable strategy against glioblastoma. In a Drosophila GB model, we have determined the post-synaptic nature of GB cells with respect to neurons, and the contribution of post-synaptic genes expressed in GB cells to tumor progression. In addition, we document the presence of intratumoral synapses between GB cells, and the functional contribution of pre-synaptic genes to GB calcium dependent activity and expansion. Finally, we explore the relevance of synaptic genes in GB cells to the lifespan reduction caused by GB advance. Our results indicate that both presynaptic and postsynaptic proteins play a role in GB progression and lethality.

Alignment between glioblastoma internal clock and environmental cues ameliorates survival in Drosophila.

Virtually every single living organism on Earth shows a circadian (i.e. "approximately a day") internal rhythm that is coordinated with planet rotation (i.e. 24 hours). External cues synchronize the central clock of the organism. Consequences of biological rhythm disruptions have been extensively studied on cancer. Still, mechanisms underlying these alterations, and how they favor tumor development remain largely unknown. Here, we show that glioblastoma-induced neurodegeneration also causes circadian alterations in Drosophila. Preventing neurodegeneration in all neurons by genetic means reestablishes normal biological rhythms. Interestingly, in early stages of tumor development, the central pacemaker lengthens its period, whereas in later stages this is severely disrupted. The re-adjustment of the external light:dark period to longer glioblastoma-induced internal rhythms delays glioblastoma progression and ameliorates associated deleterious effects, even after the tumor onset.

Mechanical control of nuclear import by Importin-7 is regulated by its dominant cargo YAP.

Mechanical forces regulate multiple essential pathways in the cell. The nuclear translocation of mechanoresponsive transcriptional regulators is an essential step for mechanotransduction. However, how mechanical forces regulate the nuclear import process is not understood. Here, we identify a highly mechanoresponsive nuclear transport receptor (NTR), Importin-7 (Imp7), that drives the nuclear import of YAP, a key regulator of mechanotransduction pathways. Unexpectedly, YAP governs the mechanoresponse of Imp7 by forming a YAP/Imp7 complex that responds to mechanical cues through the Hippo kinases MST1/2. Furthermore, YAP behaves as a dominant cargo of Imp7, restricting the Imp7 binding and the nuclear translocation of other Imp7 cargoes such as Smad3 and Erk2. Thus, the nuclear import process is an additional regulatory layer indirectly regulated by mechanical cues, which activate a preferential Imp7 cargo, YAP, which competes out other cargoes, resulting in signaling crosstalk.

Circadian Gene cry Controls Tumorigenesis through Modulation of Myc Accumulation in Glioblastoma Cells.

Glioblastoma (GB) is the most frequent malignant brain tumor among adults and currently there is no effective treatment. This aggressive tumor grows fast and spreads through the brain causing death in 15 months. GB cells display a high mutation rate and generate a heterogeneous population of tumoral cells that are genetically distinct. Thus, the contribution of genes and signaling pathways relevant for GB progression is of great relevance. We used a Drosophila model of GB that reproduces the features of human GB and describe the upregulation of the circadian gene cry in GB patients and in a Drosophila GB model. We studied the contribution of cry to the expansion of GB cells and the neurodegeneration and premature death caused by GB, and we determined that cry is required for GB progression. Moreover, we determined that the PI3K pathway regulates cry expression in GB cells, and in turn, cry is necessary and sufficient to promote Myc accumulation in GB. These results contribute to understanding the mechanisms underlying GB malignancy and lethality, and describe a novel role of Cry in GB cells.

Neural functions of small heat shock proteins.

Stress response is a cellular widespread mechanism encoded by a common protein program composed by multiple cellular factors that converge in a defense reaction to protect the cell against damage. Among many mechanisms described, heat shock proteins were proposed as universally conserved protective factors in the stress core proteome, coping with different stress stimuli through its canonical role in protein homeostasis. However, emerging evidences reveal non-canonical roles of heat shock proteins relevant for physiological and pathological conditions. Here, we review the implications of inducible heat shock proteins in the central nervous system physiology. In particular, we discuss the relevance of heat shock proteins in the maintenance of synapses, as a balanced protective mechanism in central nervous system development, pathological conditions and aging.

A novel injury paradigm in the central nervous system of adult Drosophila: molecular, cellular and functional aspects.

The mammalian central nervous system (CNS) exhibits limited regenerative capacity and the mechanisms that mediate its regeneration are not fully understood. Here, we present a novel experimental design to damage the CNS by using a contusion injury paradigm. The design of this protocol allows the study of long-term and short-term cellular responses, including those of the CNS and the immune system, and of any implications regarding functional recovery. We demonstrate for the first time that adult Drosophilamelanogaster glial cells undergo spontaneous functional recovery following crush injury. This crush injury leads to an intermediate level of functional recovery after damage, which is ideal to screen for genes that facilitate or prevent the regeneration process. Here, we validate this model and analyse the immune responses of glial cells as a central regulator of functional regeneration. Additionally, we demonstrate that glial cells and macrophages contribute to functional regeneration through mechanisms involving the Jun N-terminal kinase (JNK) pathway and the Drosophila protein Draper (Drpr), characteristic of other neural injury paradigms. We show that macrophages are recruited to the injury site and are required for functional recovery. Further, we show that the proteins Grindelwald and Drpr in Drosophila glial cells mediate activation of JNK, and that expression of drpr is dependent on JNK activation. Finally, we link neuron-glial communication and the requirement of neuronal vesicular transport to regulation of the JNK pathway and functional recovery. This article has an associated First Person interview with the first author of the paper.

JNK Pathway in CNS Pathologies.

The c-Jun N-terminal kinase (JNK) signalling pathway is a conserved response to a wide range of internal and external cellular stress signals. Beside the stress response, the JNK pathway is involved in a series of vital regulatory mechanisms during development and adulthood that are critical to maintain tissue homeostasis. These mechanisms include the regulation of apoptosis, growth, proliferation, differentiation, migration and invasion. The JNK pathway has a diverse functionality and cell-tissue specificity, and has emerged as a key player in regeneration, tumorigenesis and other pathologies. The JNK pathway is highly active in the central nervous system (CNS), and plays a central role when cells need to cope with pathophysiological insults during development and adulthood. Here, we review the implications of the JNK pathway in pathologies of the CNS. More specifically, we discuss some newly identified examples and mechanisms of JNK-driven tumor progression in glioblastoma, regeneration/repair after an injury, neurodegeneration and neuronal cell death. All these new discoveries support the central role of JNK in CNS pathologies and reinforce the idea of JNK as potential target to reduce their detrimental effects.

The haplolethality paradox of the wupA gene in Drosophila.

Haplolethals (HL) are regions of diploid genomes that in one dose are fatal for the organism. Their biological meaning is obscure because heterozygous loss-of-function mutations result in dominant lethality (DL) and, consequently, should be under strong negative selection. We report an in depth study of the HL associated to the gene wings up A (wupA). It encodes 13 transcripts (A-M) that yield 11 protein isoforms (A-K) of Troponin I (TnI). They are functionally diverse in their control of muscle contraction, cell polarity and cell proliferation. Isoform K transfers to the nucleus where it increases transcription of the cell proliferation related genes CDK2, CDK4, Rap and Rab5. The nuclear translocation of isoform K is prevented by the co-expression of A or B isoforms, which illustrates isoform interactions. The corresponding DL mutations are, either DNA rearrangements clustered towards the gene 3' end, thus affecting the genomic organization of all transcripts, or CRISPR-induced mutations in one of the two ATG sites which eliminate a subset of wupA products. The joint elimination of isoforms C, F, G and H, however, do not cause DL phenotypes. Genetically driven expression of single isoforms rescue neither DL nor any of the mutants known in the gene, suggesting that normal function requires properly regulated expression of specific combinations, rather than single, TnI isoforms. We conclude that the wupA associated HL results from the combined haploinsufficiency of a large set of TnI isoforms. The qualitative and quantitative normal expression of which, requires the chromosomal integrity of the wupA genomic region. Since all fly TnI isoforms are encoded in the same gene, its HL condition becomes unavoidable. These wupA features are comparable to those of dpp, the only other HL studied to some extent, and reveal a scenario of strict dosage dependence with implications for gene expression regulation and splitting.

Insulin signaling mediates neurodegeneration in glioma.

Cell to cell communication facilitates tissue development and physiology. Under pathological conditions, brain tumors disrupt glia-neuron communication signals that in consequence, promote tumor expansion at the expense of surrounding healthy tissue. The glioblastoma is one of the most aggressive and frequent primary brain tumors. This type of glioma expands and infiltrates into the brain, causing neuronal degeneration and neurological decay, among other symptoms. Here, we describe in a Drosophila model how glioblastoma cells produce ImpL2, an antagonist of the insulin pathway, which targets neighboring neurons and causes mitochondrial disruption as well as synapse loss, both early symptoms of neurodegeneration. Furthermore, glioblastoma progression requires insulin pathway attenuation in neurons. Restoration of neuronal insulin activity is sufficient to rescue synapse loss and to delay the premature death caused by glioma. Therefore, signals from glioblastoma to neuron emerge as a potential field of study to prevent neurodegeneration and to develop anti-tumoral strategies.

Virtual meeting, real and sound science: report of the 17th Meeting of the Spanish Society for Developmental Biology (SEBD-2020).

The Spanish Society for Developmental Biology (SEBD) organized its 17th meeting in November 2020 (herein referred to as SEBD2020). This meeting, originally programmed to take place in the city of Bilbao, was forced onto an online format due to the SARS-CoV2, COVID-19 pandemic. Although, we missed the live personal interactions and missed out on the Bilbao social scene, we were able to meet online to present our work and discuss our latest results. An overview of the activities that took place around the meeting, the different scientific sessions and the speakers involved are presented here. The pros and cons of virtual meetings are discussed.

Modeling invasion patterns in the glioblastoma battlefield.

Glioblastoma is the most aggressive tumor of the central nervous system, due to its great infiltration capacity. Understanding the mechanisms that regulate the Glioblastoma invasion front is a major challenge with preeminent potential clinical relevances. In the infiltration front, the key features of tumor dynamics relate to biochemical and biomechanical aspects, which result in the extension of cellular protrusions known as tumor microtubes. The coordination of metalloproteases expression, extracellular matrix degradation, and integrin activity emerges as a leading mechanism that facilitates Glioblastoma expansion and infiltration in uncontaminated brain regions. We propose a novel multidisciplinary approach, based on in vivo experiments in Drosophila and mathematical models, that describes the dynamics of active and inactive integrins in relation to matrix metalloprotease concentration and tumor density at the Glioblastoma invasion front. The mathematical model is based on a non-linear system of evolution equations in which the mechanisms leading chemotaxis, haptotaxis, and front dynamics compete with the movement induced by the saturated flux in porous media. This approach is able to capture the relative influences of the involved agents and reproduce the formation of patterns, which drive tumor front evolution. These patterns have the value of providing biomarker information that is related to the direction of the dynamical evolution of the front and based on static measures of proteins in several tumor samples. Furthermore, we consider in our model biomechanical elements, like the tissue porosity, as indicators of the healthy tissue resistance to tumor progression.

Cell-to-cell communication mediates glioblastoma progression in Drosophila.

Glioblastoma (GB) is the most aggressive and lethal tumour of the central nervous system (CNS). GB cells grow rapidly and display a network of projections, ultra-long tumour microtubes (TMs), that mediate cell to cell communication. GB-TMs infiltrate throughout the brain, enwrap neurons and facilitate the depletion of the signalling molecule wingless (Wg)/WNT from the neighbouring healthy neurons. GB cells establish a positive feedback loop including Wg signalling upregulation that activates cJun N-terminal kinase (JNK) pathway and matrix metalloproteases (MMPs) production, which in turn promote further TMs infiltration, GB progression and neurodegeneration. Thus, cellular and molecular signals other than primary mutations emerge as central players of GB. Using a Drosophila model of GB, we describe the temporal organisation of the main cellular events that occur in GB, including cell-to-cell interactions, neurodegeneration and TM expansion. We define the progressive activation of JNK pathway signalling in GB mediated by the receptor Grindelwald (Grnd) and activated by the ligand Eiger (Egr)/TNFα produced by surrounding healthy brain tissue. We propose that cellular interactions of GB with the healthy brain tissue precede TM expansion and conclude that non-autonomous signals facilitate GB progression. These results contribute to deciphering the complexity and versatility of these incurable tumours.

New Cellular Dimensions on Glioblastoma Progression.

Gliomas are brain tumors originated from glial cells. The most frequent form of glioma is the glioblastoma (GB). This lethal tumor is frequently originated from genetic alterations in epidermal growth factor receptor (EGFR) and PI3K pathways. Recent results suggest that signaling pathways, other than primary founder mutations, play a central role in GB progression. Some of these signals are depleted by GB cells from healthy neurons via specialized filopodia known as tumor microtubes (TMs). Here, we discuss the contribution of TMs to vampirize wingless/WNT ligand from neurons. In consequence, wingless/WNT pathway is upregulated in GB to promote tumor progression, and the reduction of these signals in neurons causes the reduction of synapse number and neurodegeneration. These processes contribute to neurological defects and premature death.

Small heat shock proteins determine synapse number and neuronal activity during development.

Environmental changes cause stress, Reactive Oxygen Species and unfolded protein accumulation which hamper synaptic activity and trigger cell death. Heat shock proteins (HSPs) assist protein refolding to maintain proteostasis and cellular integrity. Mechanisms regulating the activity of HSPs include transcription factors and posttranslational modifications that ensure a rapid response. HSPs preserve synaptic function in the nervous system upon environmental insults or pathological factors and contribute to the coupling between environmental cues and neuron control of development. We have performed a biased screening in Drosophila melanogaster searching for synaptogenic modulators among HSPs during development. We explore the role of two small-HSPs (sHSPs), sHSP23 and sHSP26 in synaptogenesis and neuronal activity. Both sHSPs immunoprecipitate together and the equilibrium between both chaperones is required for neuronal development and activity. The molecular mechanism controlling HSP23 and HSP26 accumulation in neurons relies on a novel gene (CG1561), which we name Pinkman (pkm). We propose that sHSPs and Pkm are targets to modulate the impact of stress in neurons and to prevent synapse loss.

The EGFR-TMEM167A-p53 Axis Defines the Aggressiveness of Gliomas.

Despite the high frequency of EGFR and TP53 genetic alterations in gliomas, little is known about their crosstalk during tumor progression. Here, we described a mutually exclusive distribution between mutations in these two genes. We found that wild-type p53 gliomas are more aggressive than their mutant counterparts, probably because the former accumulate amplifications and/or mutations in EGFR and show a stronger activation of this receptor. In addition, we identified a series of genes associated with vesicular trafficking of EGFR in p53 wild-type gliomas. Among these genes, TMEM167A showed the strongest implication in overall survival in this group of tumors. In agreement with this observation, inhibition of TMEM167A expression impaired the subcutaneous and the intracranial growth of wild-type p53 gliomas, regardless of the presence of EGFR mutations. In the absence of p53 mutations, TMEM167A knockdown reduced the acidification of intracellular vesicles, affecting the autophagy process and impairing EGFR trafficking and signaling. This effect was mimicked by an inhibitor of the vacuolar ATPase. We propose that the increased aggressiveness of wild-type p53 gliomas might be due to the increase in growth factor signaling activity, which depends on the regulation of vesicular trafficking by TMEM167A.

PI3K activation prevents Aß42-induced synapse loss and favors insoluble amyloid deposit formation.

Excess of Aß42 peptide is considered a hallmark of the disease. Here we express the human Aß42 peptide to assay the neuroprotective effects of PI3K in adult Drosophila melanogaster. The neuronal expression of the human peptide elicits progressive toxicity in the adult fly. The pathological traits include reduced axonal transport, synapse loss, defective climbing ability and olfactory perception, as well as lifespan reduction. The Aß42-dependent synapse decay does not involve transcriptional changes in the core synaptic protein encoding genes bruchpilot, liprin and synaptobrevin. All toxicity features, however, are suppressed by the coexpression of PI3K. Moreover, PI3K activation induces a significant increase of 6E10 and thioflavin-positive amyloid deposits. Mechanistically, we suggest that Aß42-Ser26 could be a candidate residue for direct or indirect phosphorylation by PI3K. Along with these in vivo experiments, we further analyze Aß42 toxicity and its suppression by PI3K activation in in vitro assays with SH-SY5Y human neuroblastoma cell cultures, where Aß42 aggregation into large insoluble deposits is reproduced. Finally, we show that the Aß42 toxicity syndrome includes the transcriptional shut down of PI3K expression. Taken together, these results uncover a potential novel pharmacological strategy against this disease through the restoration of PI3K activity.

Glioblastoma cells vampirize WNT from neurons and trigger a JNK/MMP signaling loop that enhances glioblastoma progression and neurodegeneration.

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, ß-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.

Cytonemes, Their Formation, Regulation, and Roles in Signaling and Communication in Tumorigenesis.

Increasing evidence during the past two decades shows that cells interconnect and communicate through cytonemes. These cytoskeleton-driven extensions of specialized membrane territories are involved in cell-cell signaling in development, patterning, and differentiation, but also in the maintenance of adult tissue homeostasis, tissue regeneration, and cancer. Brain tumor cells in glioblastoma extend ultralong membrane protrusions (named tumor microtubes, TMs), which contribute to invasion, proliferation, radioresistance, and tumor progression. Here we review the mechanisms underlying cytoneme formation, regulation, and their roles in cell signaling and communication in epithelial cells and other cell types. Furthermore, we discuss the recent discovery of glial cytonemes in the Drosophila glial cells that alter Wingless (Wg)/Frizzled (Fz) signaling between glia and neurons. Research on cytoneme formation, maintenance, and cell signaling mechanisms will help to better understand not only physiological developmental processes and tissue homeostasis but also cancer progression.

Troponin-I mediates the localization of selected apico-basal cell polarity signaling proteins.

Beyond its role in muscle contraction, Drosophila Troponin I (TnI; also known as Wings up A) is expressed in epithelial cells where it controls proliferation. TnI traffics between nucleus and cytoplasm through a sumoylation-dependent mechanism. We address here the role of TnI in the cytoplasm. TnI accumulates apically in epidermal cells and neuroblasts. TnI co-immunoprecipitates with Bazooka (also known as Par3) and Discs large (Dlg1, hereafter Dlg), two apico-basal polarity components. TnI depletion causes Baz and Dlg mislocalization; by contrast, the basolateral localization of Scribbled is not altered. In neuroblasts, TnI contributes to the polar localization of Miranda, while non-polar Dlg localization is not affected. Vertebrate phosphoinositide 3-kinase (PI3K) contributes to the apico-basal polarity of epithelia, but we find that Drosophila PI3K depletion alters neither the apical localization of TnI or Bazooka, nor the basal localization of Dlg. Nevertheless, overexpressing PI3K prevents the defects seen upon TnI depletion. TnI loss-of-function disrupts cytoskeletal ß-Catenin, E-Cadherin and γ-Tubulin, and causes an increase in DNA damage, as revealed by analyzing γH2Av. We have previously shown that TnI depletion leads to apoptosis that can be suppressed by upregulating Sparc or downregulating Dronc. However, TnI-depleted cells expressing Sparc or downregulating Dronc, as well as those expressing p35 (also known as Cdk5α), that do not undergo apoptosis, still show DNA damage. This indicates that DNA damage is mechanistically independent of apoptosis induction. Thus, TnI binds certain apico-basal polarity signaling proteins in a cell type-dependent context, and this unveils a previously unsuspected diversity of mechanisms to allocate cell polarity factors.