ABSTRACT
Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM-associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient-derived GBM stem cell cultures and combined single-cell RNA sequencing of GAM-GBM co-cultures and real-time in vivo monitoring of GAM-GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM-induced GAM polarization and the ability to attract and activate GAMs-features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM-GBM interactions can be studied in a clinically relevant way using co-cultures and avatar models, while offering new opportunities to identify promising immune-modulating targets.
Subject(s)
Brain Neoplasms , Glioblastoma , Animals , Humans , Glioblastoma/pathology , Zebrafish , Galectin 1/genetics , Galectin 1/metabolism , Galectin 1/therapeutic use , Cell Line, Tumor , Macrophages/metabolism , Brain Neoplasms/pathology , Tumor Microenvironment/geneticsABSTRACT
BACKGROUND: Functional profiling of freshly isolated glioblastoma (GBM) cells is being evaluated as a next-generation method for precision oncology. While promising, its success largely depends on the method to evaluate treatment activity which requires sufficient resolution and specificity. METHODS: Here, we describe the 'precision oncology by single-cell profiling using ex vivo readouts of functionality' (PROSPERO) assay to evaluate the intrinsic susceptibility of high-grade brain tumor cells to respond to therapy. Different from other assays, PROSPERO extends beyond life/death screening by rapidly evaluating acute molecular drug responses at single-cell resolution. RESULTS: The PROSPERO assay was developed by correlating short-term single-cell molecular signatures using mass cytometry by time-of-flight (CyTOF) to long-term cytotoxicity readouts in representative patient-derived glioblastoma cell cultures (n = 14) that were exposed to radiotherapy and the small-molecule p53/MDM2 inhibitor AMG232. The predictive model was subsequently projected to evaluate drug activity in freshly resected GBM samples from patients (n = 34). Here, PROSPERO revealed an overall limited capacity of tumor cells to respond to therapy, as reflected by the inability to induce key molecular markers upon ex vivo treatment exposure, while retaining proliferative capacity, insights that were validated in patient-derived xenograft (PDX) models. This approach also allowed the investigation of cellular plasticity, which in PDCLs highlighted therapy-induced proneural-to-mesenchymal (PMT) transitions, while in patients' samples this was more heterogeneous. CONCLUSION: PROSPERO provides a precise way to evaluate therapy efficacy by measuring molecular drug responses using specific biomarker changes in freshly resected brain tumor samples, in addition to providing key functional insights in cellular behavior, which may ultimately complement standard, clinical biomarker evaluations.
Subject(s)
Antineoplastic Agents , Brain Neoplasms , Glioblastoma , Humans , Glioblastoma/pathology , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Precision Medicine , Antineoplastic Agents/therapeutic use , Xenograft Model Antitumor Assays , Cell Line, TumorABSTRACT
In malignant cancer, excessive amounts of mutant p53 often lead to its aggregation, a feature that was recently identified as druggable. Here, we describe that induction of a heat shock-related stress response mediated by Foldlin, a small-molecule tool compound, reduces the protein levels of misfolded/aggregated mutant p53, while contact mutants or wild-type p53 remain largely unaffected. Foldlin also prevented the formation of stress-induced p53 nuclear inclusion bodies. Despite our inability to identify a specific molecular target, Foldlin also reduced protein levels of aggregating SOD1 variants. Finally, by screening a library of 778 FDA-approved compounds for their ability to reduce misfolded mutant p53, we identified the proteasome inhibitor Bortezomib with similar cellular effects as Foldlin. Overall, the induction of a cellular heat shock response seems to be an effective strategy to deal with pathological protein aggregation. It remains to be seen however, how this strategy can be translated to a clinical setting.
Subject(s)
Protein Folding , Tumor Suppressor Protein p53 , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Proteasome Inhibitors/pharmacology , Heat-Shock Response , Bortezomib/pharmacologyABSTRACT
The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Receptors, Wnt/metabolism , Amino Acid Sequence , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/genetics , Animals , Brain/cytology , Cells, Cultured , Cloning, Molecular , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster , Gene Deletion , Gene Expression Regulation/physiology , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mushroom Bodies/cytology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Protein Transport , Receptors, Wnt/genetics , Signal TransductionABSTRACT
Size trade-offs of visual versus olfactory organs is a pervasive feature of animal evolution. This could result from genetic or functional constraints. We demonstrate that head sensory organ size trade-offs in Drosophila are genetically encoded and arise through differential subdivision of the head primordium into visual versus non-visual fields. We discover that changes in the temporal regulation of the highly conserved eyeless/Pax6 gene expression during development is a conserved mechanism for sensory trade-offs within and between Drosophila species. We identify a natural single nucleotide polymorphism in the cis-regulatory region of eyeless in a binding site of its repressor Cut that is sufficient to alter its temporal regulation and eye size. Because eyeless/Pax6 is a conserved regulator of head sensory placode subdivision, we propose that its temporal regulation is key to define the relative size of head sensory organs.
Subject(s)
Biological Evolution , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental , Sense Organs/metabolism , Animals , Binding Sites , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Enhancer Elements, Genetic/genetics , Eye/anatomy & histology , Eye/metabolism , Female , Geography , Head , Nucleotides/genetics , Organ Size/genetics , Polymorphism, Single Nucleotide/genetics , Time FactorsABSTRACT
Isolation profoundly influences social behavior in all animals. In humans, isolation has serious effects on health. Drosophila melanogaster is a powerful model to study small-scale, temporally-transient social behavior. However, longer-term analysis of large groups of flies is hampered by the lack of effective and reliable tools. We built a new imaging arena and improved the existing tracking algorithm to reliably follow a large number of flies simultaneously. Next, based on the automatic classification of touch and graph-based social network analysis, we designed an algorithm to quantify changes in the social network in response to prior social isolation. We observed that isolation significantly and swiftly enhanced individual and local social network parameters depicting near-neighbor relationships. We explored the genome-wide molecular correlates of these behavioral changes and found that whereas behavior changed throughout the six days of isolation, gene expression alterations occurred largely on day one. These changes occurred mostly in metabolic genes, and we verified the metabolic changes by showing an increase of lipid content in isolated flies. In summary, we describe a highly reliable tracking and analysis pipeline for large groups of flies that we use to unravel the behavioral, molecular and physiological impact of isolation on social network dynamics in Drosophila.
Subject(s)
Behavior, Animal/physiology , Population Surveillance/methods , Social Isolation/psychology , Algorithms , Animals , Computers , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Interpersonal Relations , Social Behavior , SoftwareABSTRACT
The importance of producing the correct numbers of neurons during development is illustrated by both evolutionary enhancement of cognitive capacities in larger brains, and developmental disorders of brain size. In humans, increased neuronal numbers during development is speculated to partly derive from a unique subtype of neural stem cells (NSCs) that undergo a phase of expansion through symmetric self-amplifying divisions before generating neurons. Symmetric amplification also appears to underlie adult neural stem maintenance in the mouse. However, the mechanisms regulating this behavior are unclear. We report the discovery of self-amplifying NSCs in Drosophila and show that they arise by a spatiotemporal conversion of classical self-renewing NSCs. This conversion is regulated by a temporal transition in the expression of proneural transcription factors prior to cell division. We find a causal link between stem cell self-amplification and increased neuronal numbers. We further show that the temporal transcriptional switch controls both stem cell division and subsequent neuronal differentiation.
Subject(s)
Cell Differentiation , Cell Proliferation , Drosophila melanogaster/growth & development , Neural Stem Cells/cytology , Neurogenesis/physiology , Neurons/cytology , Animals , Cell Count , Cell Self Renewal , Cells, Cultured , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Female , Male , Neural Stem Cells/metabolism , Neurons/metabolism , Signal Transduction , Transcription, GeneticABSTRACT
Animals are characterized by a set of highly conserved developmental regulators. Changes in the cis-regulatory elements of these regulators are thought to constitute the major driver of morphological evolution. However, the role of coding sequence evolution remains unresolved. To address this question, we used the Atonal family of proneural transcription factors as a model. Drosophila atonal coding sequence was endogenously replaced with that of atonal homologues (ATHs) at key phylogenetic positions, non-ATH proneural genes, and the closest homologue to ancestral proneural genes. ATHs and the ancestral-like coding sequences rescued sensory organ fate in atonal mutants, in contrast to non-ATHs. Surprisingly, different ATH factors displayed different levels of proneural activity as reflected by the number and functionality of sense organs. This proneural potency gradient correlated directly with ATH protein stability, including in response to Notch signaling, independently of mRNA levels or codon usage. This establishes a distinct and ancient function for ATHs and demonstrates that coding sequence evolution can underlie quantitative variation in sensory development and function.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Drosophila Proteins/genetics , Drosophila/embryology , Nerve Tissue Proteins/genetics , Transcription, Genetic , Animal Structures/embryology , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Drosophila/metabolism , Drosophila Proteins/metabolism , Morphogenesis , Mutant Proteins/genetics , Mutant Proteins/metabolism , Nerve Tissue Proteins/metabolism , Recombination, GeneticABSTRACT
Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3'-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism.
ABSTRACT
The axonal wiring molecule Slit and its Round-About (Robo) receptors are conserved regulators of nerve cord patterning. Robo receptors also contribute to wiring brain circuits. Whether molecular mechanisms regulating these signals are modified to fit more complex brain wiring processes is unclear. We investigated the role of Slit and Robo receptors in wiring Drosophila higher-order brain circuits and identified differences in the cellular and molecular mechanisms of Robo/Slit function. First, we find that signaling by Robo receptors in the brain is regulated by the Receptor Protein Tyrosine Phosphatase RPTP69d. RPTP69d increases membrane availability of Robo3 without affecting its phosphorylation state. Second, we detect no midline localization of Slit during brain development. Instead, Slit is enriched in the mushroom body, a neuronal structure covering large areas of the brain. Thus, a divergent molecular mechanism regulates neuronal circuit wiring in the Drosophila brain, partly in response to signals from the mushroom body.
Subject(s)
Brain/metabolism , Drosophila Proteins/metabolism , Nerve Net/metabolism , Nerve Tissue Proteins/metabolism , Neuropil/metabolism , Receptor-Like Protein Tyrosine Phosphatases/metabolism , Receptors, Immunologic/metabolism , Signal Transduction , Animals , Axons/metabolism , Cell Membrane/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Epistasis, Genetic , Gene Expression Regulation, Developmental , HEK293 Cells , Humans , Larva/metabolism , Multiprotein Complexes/metabolism , Mushroom Bodies/metabolism , Nerve Tissue Proteins/genetics , PhenotypeABSTRACT
The neurogenin (Ngn) transcription factors control early neurogenesis and neurite outgrowth in mammalian cortex. In contrast to their proneural activity, their function in neurite growth is poorly understood. Drosophila has a single predicted Ngn homolog, Tap, of unknown function. Here we show that Tap is not a proneural protein in Drosophila but is required for proper axonal growth and guidance of neurons of the mushroom body, a neuropile required for associative learning and memory. Genetic and expression analyses suggest that Tap inhibits excessive axonal growth by fine regulation of the levels of the Wnt signaling adaptor protein Dishevelled.
Subject(s)
Cell Polarity/physiology , Drosophila Proteins/metabolism , Neuropeptides/metabolism , Transcription Factors/metabolism , Wnt Signaling Pathway/physiology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Axon Guidance/genetics , Axon Guidance/physiology , Axons/metabolism , Cell Polarity/genetics , Drosophila , Drosophila Proteins/genetics , Mushroom Bodies/metabolism , Neuropeptides/genetics , Protein Binding , Transcription Factors/genetics , Wnt Signaling Pathway/geneticsABSTRACT
Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. Here, we discover a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, we show that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/genetics , Neurogenesis , Amino Acid Sequence , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Drosophila , Drosophila Proteins , Eye/growth & development , Eye/ultrastructure , Imaginal Discs/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Phosphorylation , Retina/growth & development , Sequence AlignmentABSTRACT
Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.
Subject(s)
Cell Nucleus , Fragile X Mental Retardation Protein , Fragile X Syndrome , Mutation , Nuclear Localization Signals , Trinucleotide Repeat Expansion , Animals , Cell Line, Transformed , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cell Nucleus/pathology , Drosophila melanogaster , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/genetics , Fragile X Syndrome/metabolism , Fragile X Syndrome/pathology , Humans , Male , Mice , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , Protein Structure, Tertiary , Protein Transport/geneticsABSTRACT
Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αß neurons and, in particular, is required cell-autonomously for the ß-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.
Subject(s)
Amyloid beta-Protein Precursor/genetics , Drosophila/metabolism , Signal Transduction , Wnt Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Cell Polarity , Dishevelled Proteins , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , HEK293 Cells , Humans , Mushroom Bodies/cytology , Mushroom Bodies/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolismABSTRACT
A comprehensive systems-level understanding of developmental programs requires the mapping of the underlying gene regulatory networks. While significant progress has been made in mapping a few such networks, almost all gene regulatory networks underlying cell-fate specification remain unknown and their discovery is significantly hampered by the paucity of generalized, in vivo validated tools of target gene and functional enhancer discovery. We combined genetic transcriptome perturbations and comprehensive computational analyses to identify a large cohort of target genes of the proneural and tumor suppressor factor Atonal, which specifies the switch from undifferentiated pluripotent cells to R8 photoreceptor neurons during larval development. Extensive in vivo validations of the predicted targets for the proneural factor Atonal demonstrate a 50% success rate of bona fide targets. Furthermore we show that these enhancers are functionally conserved by cloning orthologous enhancers from Drosophila ananassae and D. virilis in D. melanogaster. Finally, to investigate cis-regulatory cross-talk between Ato and other retinal differentiation transcription factors (TFs), we performed motif analyses and independent target predictions for Eyeless, Senseless, Suppressor of Hairless, Rough, and Glass. Our analyses show that cisTargetX identifies the correct motif from a set of coexpressed genes and accurately predicts target genes of individual TFs. The validated set of novel Ato targets exhibit functional enrichment of signaling molecules and a subset is predicted to be coregulated by other TFs within the retinal gene regulatory network.