One of the main limitations to anticancer radiotherapy lies in irreversible damage to healthy tissues located within the radiation field. "FLASH" irradiation at very high dose-rate is a new treatment ...modality that has been reported to specifically spare normal tissue from late radiation-induced toxicity in animal models and therefore could be a promising strategy to reduce treatment toxicity.
Lung responses to FLASH irradiation were investigated by qPCR, single-cell RNA sequencing (sc-RNA-Seq), and histologic methods during the acute wound healing phase as well as at late stages using C57BL/6J wild-type and Terc
mice exposed to bilateral thorax irradiation as well as human lung cells grown
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studies gave evidence of a reduced level of DNA damage and induced lethality at the advantage of FLASH. In mouse lung, sc-RNA-seq and the monitoring of proliferating cells revealed that FLASH minimized the induction of proinflammatory genes and reduced the proliferation of progenitor cells after injury. At late stages, FLASH-irradiated lungs presented less persistent DNA damage and senescent cells than after CONV exposure, suggesting a higher potential for lung regeneration with FLASH. Consistent with this hypothesis, the beneficial effect of FLASH was lost in Terc
mice harboring critically short telomeres and lack of telomerase activity.
The results suggest that, compared with conventional radiotherapy, FLASH minimizes DNA damage in normal cells, spares lung progenitor cells from excessive damage, and reduces the risk of replicative senescence.
Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human ...pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo-electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.
Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative ...breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.
The current consensus recognizes four main medulloblastoma subgroups (wingless, Sonic hedgehog, group 3 and group 4). While medulloblastoma subgroups have been characterized extensively at the ...(epi-)genomic and transcriptomic levels, the proteome and phosphoproteome landscape remain to be comprehensively elucidated. Using quantitative (phospho)-proteomics in primary human medulloblastomas, we unravel distinct posttranscriptional regulation leading to highly divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas. Specifically, proteomic and phosphoproteomic analyses identify aberrant ERBB4-SRC signaling in group 4. Hence, enforced expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 medulloblastoma. Therefore, our integrative proteogenomics approach unveils an oncogenic pathway and potential therapeutic vulnerability in the most common medulloblastoma subgroup.
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•Highly divergent posttranscriptional pathway regulation in MB subgroups•Phosphoproteomic profiles reveal specific kinase activity in MB subgroups•Identification of aberrant ERBB4-SRC signaling as a hallmark of group 4 MBs•Over expression of activated SRC in the developing cerebellum induces MB
Using proteomic analyses, Forget et al. unravel divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas (MB) and identify aberrant ERBB4-SRC signaling in group 4. Expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 MB.
Radiation Induced Lung Injury (RILI) is one of the main limiting factors of thorax irradiation, which can induce acute pneumonitis as well as pulmonary fibrosis, the latter being a life-threatening ...condition. The order of cellular and molecular events in the progression towards fibrosis is key to the physiopathogenesis of the disease, yet their coordination in space and time remains largely unexplored. Here, we present an interactive murine single cell atlas of the lung response to irradiation, generated from C57BL6/J female mice. This tool opens the door for exploration of the spatio-temporal dynamics of the mechanisms that lead to radiation-induced pulmonary fibrosis. It depicts with unprecedented detail cell type-specific radiation-induced responses associated with either lung regeneration or the failure thereof. A better understanding of the mechanisms leading to lung fibrosis will help finding new therapeutic options that could improve patients' quality of life.
Sperm cells are highly specialized mammalian cells, and their biogenesis requires unique intracellular structures. Perturbation of spermatogenesis often leads to male infertility. Here, we assess the ...role of a post-translational modification of tubulin, glutamylation, in spermatogenesis. We show that mice lacking the tubulin deglutamylase CCP5 (also known as AGBL5) do not form functional sperm. In these mice, spermatids accumulate polyglutamylated tubulin, accompanied by the occurrence of disorganized microtubule arrays, in particular in the sperm manchette. Spermatids further fail to re-arrange their intracellular space and accumulate organelles and cytosol, while nuclei condense normally. Strikingly, spermatids lacking CCP5 show supernumerary centrioles, suggesting that glutamylation could control centriole duplication. We show that most of these observed defects are also present in mice in which CCP5 is deleted only in the male germ line, strongly suggesting that they are germ-cell autonomous. Our findings reveal that polyglutamylation is, beyond its known importance for sperm flagella, an essential regulator of several microtubule-based functions during spermatogenesis. This makes enzymes involved in glutamylation prime candidates for being genes involved in male sterility.
During cerebellar development, granule neuron progenitors (GNPs) proliferate by transducing Sonic Hedgehog (SHH) signaling via the primary cilium. Precise regulation of ciliogenesis, thus, ensures ...proper GNP pool expansion. Here, we report that Atoh1, a transcription factor required for GNPs formation, controls the presence of primary cilia, maintaining GNPs responsiveness to SHH. Loss of primary cilia abolishes the ability of Atoh1 to keep GNPs in a proliferative state. Mechanistically, Atoh1 promotes ciliogenesis by transcriptionally regulating Cep131, which facilitates centriolar satellite (CS) clustering to the basal body. Importantly, ectopic expression of Cep131 counteracts the effects of Atoh1 loss in GNPs by restoring proper localization of CS and ciliogenesis. This Atoh1-CS-primary cilium-SHH pro-proliferative pathway is also conserved in SHH-type medulloblastoma, a pediatric brain tumor arising from the GNPs. Together, our data reveal how Atoh1 modulates the primary cilium to regulate GNPs development.
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•Atoh1 controls the presence of primary cilia in cerebellar granule neuron progenitors•Atoh1 requires the primary cilium to maintain the pool of proliferating progenitors•Atoh1 controls ciliogenesis by shaping centriolar satellites via its target Cep131
Chang et al. demonstrate how Atoh1 controls granule neuron progenitor proliferation during postnatal cerebellar development. By transcriptionally regulating Cep131, Atoh1 maintains the presence of the primary cilium, an organelle required for SHH signal transduction. This mechanism is also conserved in SHH medulloblastoma, a malignant pediatric brain tumor arising from the cerebellum.
Medulloblastoma (MB) is a pediatric tumor of the cerebellum divided into four groups. Group 3 is of bad prognosis and remains poorly characterized. While the current treatment involving surgery, ...radiotherapy, and chemotherapy often fails, no alternative therapy is yet available. Few recurrent genomic alterations that can be therapeutically targeted have been identified. Amplifications of receptors of the TGFβ/Activin pathway occur at very low frequency in Group 3 MB. However, neither their functional relevance nor activation of the downstream signaling pathway has been studied. We showed that this pathway is activated in Group 3 MB with some samples showing a very strong activation. Beside genetic alterations, we demonstrated that an ActivinB autocrine stimulation is responsible for pathway activation in a subset of Group 3 MB characterized by high PMEPA1 levels. Importantly, Galunisertib, a kinase inhibitor of the cognate receptors currently tested in clinical trials for Glioblastoma patients, showed efficacy on orthotopically grafted MB‐PDX. Our data demonstrate that the TGFβ/Activin pathway is active in a subset of Group 3 MB and can be therapeutically targeted.
Synopsis
TGFβ/Activin signaling is activated in a subset of Group 3 medulloblastoma, through an ActivinB autocrine loop. ActivinB sustains tumor cell growth by inducing the expression of the Smad target gene PMEPA1. TGFβ/Activin pathway can be therapeutically targeted with Galunisertib in this G3 subgroup.
A subset of G3 medulloblastoma tumors displays high TGFβ/Activin pathway activation.
An ActivinB autocrine loop is responsible for TGFβ/Activin pathway activation in G3 medulloblastoma.
G3 medulloblastoma with strong TGFβ/Activin pathway activation are characterized by a high level of PMEPA1 expression.
Galunisertib treatment is of potential therapeutic interest for G3 medulloblastoma showing strong TGFβ/Activin pathway activation.
TGFβ/Activin signaling is activated in a subset of Group 3 medulloblastoma, through an ActivinB autocrine loop. ActivinB sustains tumor cell growth by inducing the expression of the Smad target gene PMEPA1. TGFβ/Activin pathway can be therapeutically targeted with Galunisertib in this G3 subgroup.
Posttranslational modifications of tubulin are emerging regulators of microtubule functions. We have shown earlier that upregulated polyglutamylation is linked to rapid degeneration of Purkinje cells ...in mice with a mutation in the deglutamylating enzyme CCP1. How polyglutamylation leads to degeneration, whether it affects multiple neuron types, or which physiological processes it regulates in healthy neurons has remained unknown. Here, we demonstrate that excessive polyglutamylation induces neurodegeneration in a cell‐autonomous manner and can occur in many parts of the central nervous system. Degeneration of selected neurons in CCP1‐deficient mice can be fully rescued by simultaneous knockout of the counteracting polyglutamylase TTLL1. Excessive polyglutamylation reduces the efficiency of neuronal transport in cultured hippocampal neurons, suggesting that impaired cargo transport plays an important role in the observed degenerative phenotypes. We thus establish polyglutamylation as a cell‐autonomous mechanism for neurodegeneration that might be therapeutically accessible through manipulation of the enzymes that control this posttranslational modification.
Synopsis
Excessive accumulation of polyglutamylation, a posttranslational modification of the neuronal microtubule cytoskeleton, leads to degeneration of a variety of neurons in mouse brains, and perturbs axonal transport.
Removal of two deglutamylating enzymes in mouse brain leads to hyperglutamylation due to unopposed glutamylase activity in the entire brain.
Hyperglutamylation can cause the degeneration of various neurons in the mouse brain.
Concomitant deletion of the main brain glutamylase can prevent hyperglutamylation‐induced neurodegeneration in a cell‐autonomous manner.
The microtubule‐severing enzyme spastin is not responsible for hyperglutamylation‐induced neurodegeneration.
Axonal transport is perturbed in neurons with tubulin hyperglutamylation.
Neurodegeneration in mice lacking the tubulin‐deglutamylating enzyme CCP1 occurs in a cell‐autonomous manner and in various parts of the central nervous system.
Tubulin polyglutamylation is a post‐translational modification of the microtubule cytoskeleton, which is generated by a variety of enzymes with different specificities. The “tubulin code” hypothesis ...predicts that modifications generated by specific enzymes selectively control microtubule functions. Our recent finding that excessive accumulation of polyglutamylation in neurons causes their degeneration and perturbs axonal transport provides an opportunity for testing this hypothesis. By developing novel mouse models and a new glutamylation‐specific antibody, we demonstrate here that the glutamylases TTLL1 and TTLL7 generate unique and distinct glutamylation patterns on neuronal microtubules. We find that under physiological conditions, TTLL1 polyglutamylates α‐tubulin, while TTLL7 modifies β‐tubulin. TTLL1, but not TTLL7, catalyses the excessive hyperglutamylation found in mice lacking the deglutamylase CCP1. Consequently, deletion of TTLL1, but not of TTLL7, prevents degeneration of Purkinje cells and of myelinated axons in peripheral nerves in these mice. Moreover, loss of TTLL1 leads to increased mitochondria motility in neurons, while loss of TTLL7 has no such effect. By revealing how specific patterns of tubulin glutamylation, generated by distinct enzymes, translate into specific physiological and pathological readouts, we demonstrate the relevance of the tubulin code for homeostasis.
SYNOPSIS
Polyglutamylation is a posttranslational modification of tubulin that is highly enriched in neurons. Here we demonstrate that two neuronal polyglutamylases, TTLL1 and TTLL7, have distinct enzymatic activities, which generate unique patterns of polyglutamylation in vivo. We find that TTLL1, but not TTLL7 affects mitochondria transport and neuronal survival, in both central and peripheral nervous system.
TTLL1 polyglutamylates α‐tubulin, while TTLL7 modifies β‐tubulin in vivo.
In the absence of the deglutamylase CCP1, excessive polyglutamylation leading to neurodegeneration is generated by TTLL1, but not by TTLL7.
Degeneration of neurons in both, central and peripheral nervous system, can be avoided by inactivating TTLL1, but not TTLL7.
Polyglutamylation generated by TTLL1, but not by TTLL7 affects mitochondria mobility in axons of hippocampal neurons.
New mouse models and modification‐specific antibodies reveals that tubulin glutamylases TTLL1 and TTLL7 generate unique glutamylation patterns on neuronal microtubules, which regulate mitochondria motility and neuronal survival.
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