Year of publication 2020
Experimental and Preclinical Tools to Explore the Main Neurological Impacts of Brain Irradiation: Current Insights and PerspectivesBrain Tumors : 158 : 239-261 : DOI : 10.1007/978-1-0716-0856-2_11 Learn more
Radiation therapy is a powerful tool in the treatment of primary and metastatic cancers of the brain. However, brain tissue tolerance is limited, and radiation doses must be tailored to minimize deleterious effects on the nervous system. Due to improved treatments, including radiotherapy techniques, many patients with brain tumors survive longer, but they experience late effects of radiotherapy, especially cognitive decline, for which no efficient treatment is currently available. Improving the prevention and treatment of radiation-induced neurological defects first needs to better characterize radiation injuries in brain cells and tissues. Rodent models have been widely used for this.
Here, observations from patients will be reviewed briefly as an introduction, mainly regarding clinical cognitive defects and anatomical alterations using magnetic resonance imaging (MRI). This limited descriptive clinical knowledge addresses many questions that arise in preclinical models regarding understanding the mechanism of radiation-induced brain dysfunction. From this perspective, we next present methods to characterize radiation-induced neurogenesis alterations in adult mice and then detail how MRI could be used as a powerful tool to explore these alterations.Fold up
A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD+Scientific Reports : 10 : 13907 : DOI : 10.1038/s41598-020-70874-6 Learn more
Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.Fold up
Two-Photon Emissive Dyes Based on Push–Pull Purines Derivatives: Toward the Development of New Photoluminescence BioprobesThe Journal of Physical Chemistry C : 124 : 12185-12864 : DOI : 10.1021/acs.jpcc.0c01859 Learn more
Fluorescent organic molecules have received great attention due to their largest applications, for example, in DNA and RNA spectroscopies studies, development of new photoluminescence bioprobes, and applications in fluorescence spectroscopy. In specific, purine base analog molecules present high fluorescence quantum yields and significant Stokes shift. Furthermore, the addition of push–pull structures at the purine core could increase the photoluminescence properties, making candidates for photoluminescence bioprobes. To consider this, a complete spectroscopic study was performed on nine push–pull purines, distinguished by different push–pull structures. In specific, for this research, the two-photon absorption (2PA) study showed that the compounds present induced two-photon fluorescence at the therapeutic window, desired for fluorescence microscopy. The brightness property was evaluated, indicating that all chromospheres are fluorescent by a 2PA process. Additionally, ultrafast transient absorption was performed to elucidate contribution of the excited states on the 2PA spectra, and quantum chemistry calculations were performed to corroborate the experimental results.Fold up
Intracellular location matters: rationalization of the anti-inflammatory activity of a manganese (II) superoxide dismutase mimic complexChem. Commun. : 56 : 7885-7888 : DOI : 10.1039/D0CC03398G Learn more
A conjugate of a Mn-based superoxide dismutase mimic with a Re-based multimodal probe was studied in a cellular model of oxidative stress. Its speciation was questioned using Re and Mn X-fluorescence. Its distribution were compared to its unconjugated analogue. Interestingly, their similar activities mirror their similar concentrations in mitochondria.Fold up
Low retinal toxicity of intravitreal carboplatin associated with good retinal tumor control in transgenic murine retinoblastoma.Clinical & experimental ophthalmology : 48 : 500-511 : DOI : 10.1111/ceo.13711 Learn more
Background: Retinoblastoma is a rare intraocular malignancy in children. Current treatments have many adverse effects. New therapeutic approaches like intravitreal injections of chemotherapies are currently being developed but their toxicities need to be evaluated on animal models. This study compares the efficacy and toxicity of intravitreal melphalan, topotecan and carboplatin, alone or in combination (sequential administration), in the LHBetaTag retinoblastoma mice. Methods: Mice were divided into 9 groups: control, carboplatin 1.5 and 4μg, melphalan 0.1 and 1μg, topotecan 0.1 and 1μg, carboplatin 4μg/ topotecan 0.1μg and melphalan 1μg/ topotecan 0.1μg. The follow‐up was performed using fundus imaging and optical coherence tomography combined with histopathological analysis. Absence of tumor and presence of calcified tumors were the criteria for therapeutic response assessment. Ocular complications were assessed after 4 weekly injections. Retinal toxicity was defined by the decrease of retinal thickness and of the number of retinal layers. Results: Topotecan was inactive on retinal tumors. Melphalan (1μg) led to a complete tumor control in 91.7% of eyes. Carboplatin strongly decreased the tumor burden (85.7‐93.8% of eyes without retinal tumor). The intravitreal injection itself led to ocular complications (25% of media opacities and 45.7% of retinal detachment). Only melphalan at 1μg showed a strong retinal toxicity. The two combinations showed a good efficacy in reducing the number of eyes with retinal tumors with a reduced retinal toxicity. Conclusions: This preclinical study suggests that intravitreal injection of carboplatin has a low toxicity and could be evaluated in clinical practice to treat patients suffering from retinoblastoma.
Background: Retinoblastoma is a rare intraocular malignancy in children. Current treatments have many adverse effects. New therapeutic approaches like intravitreal injections of chemotherapies are currently being developed but their toxicities need to be evaluated on animal models. This study compares the efficacy and toxicity of intravitreal melphalan, topotecan and carboplatin, alone or in combination (sequential administration), in the LHBetaTag retinoblastoma mice.
Methods: Mice were divided into 9 groups: control, carboplatin 1.5 and 4μg, melphalan 0.1 and 1μg, topotecan 0.1 and 1μg, carboplatin 4μg/ topotecan 0.1μg and melphalan 1μg/ topotecan 0.1μg. The follow‐up was performed using fundus imaging and optical coherence tomography combined with histopathological analysis. Absence of tumor and presence of calcified tumors were the criteria for therapeutic response assessment. Ocular complications were assessed after 4 weekly injections. Retinal toxicity was defined by the decrease of retinal thickness and of the number of retinal layers.
Results: Topotecan was inactive on retinal tumors. Melphalan (1μg) led to a complete tumor control in 91.7% of eyes. Carboplatin strongly decreased the tumor burden (85.7‐93.8% of eyes without retinal tumor). The intravitreal injection itself led to ocular complications (25% of media opacities and 45.7% of retinal detachment). Only melphalan at 1μg showed a strong retinal toxicity. The two combinations showed a good efficacy in reducing the number of eyes with retinal tumors with a reduced retinal toxicity.
Conclusions: This preclinical study suggests that intravitreal injection of carboplatin has a low toxicity and could be evaluated in clinical practice to treat patients suffering from retinoblastoma.
Interplay between cellular uptake, intracellular localization and the cell death mechanism in triphenylamine-mediated photoinduced cell deathScientific Reports : 10 : 6881 : DOI : 10.1038/s41598-020-63991-9 Learn more
Triphenylamines (TPAs) were previously shown to trigger cell death under prolonged one- or two-photon illumination. Their initial subcellular localization, before prolonged illumination, is exclusively cytoplasmic and they translocate to the nucleus upon photoactivation. However, depending on their structure, they display significant differences in terms of precise initial localization and subsequent photoinduced cell death mechanism. Here, we investigated the structural features of TPAs that influence cell death by studying a series of molecules differing by the number and chemical nature of vinyl branches. All compounds triggered cell death upon one-photon excitation, however to different extents, the nature of the electron acceptor group being determinant for the overall cell death efficiency. Photobleaching susceptibility was also an important parameter for discriminating efficient/inefficient compounds in two-photon experiments. Furthermore, the number of branches, but not their chemical nature, was crucial for determining the cellular uptake mechanism of TPAs and their intracellular fate. The uptake of all TPAs is an active endocytic process but two- and three-branch compounds are taken up via distinct endocytosis pathways, clathrin-dependent or -independent (predominantly caveolae-dependent), respectively. Two-branch TPAs preferentially target mitochondria and photoinduce both apoptosis and a proper necrotic process, whereas three-branch TPAs preferentially target late endosomes and photoinduce apoptosis only.Fold up
Harnessing intrinsic fluorescence for typing of secondary structures of DNANucleic Acids Research : 48 : e61 : DOI : 10.1093/nar/gkaa257 Learn more
High-throughput investigation of structural diversity of nucleic acids is hampered by the lack of suitable label-free methods, combining fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of suitability of this phenomenon for tracking conformational changes of DNA, we examined steady-state emission spectra of an 89-membered set of oligonucleotides with reported conformation (G-quadruplexes (G4s), i-motifs, single- and double-strands) by means of multivariate analysis. Principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, without discrimination between single- and double-stranded structures. Linear discriminant analysis was exploited for the assessment of novel sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labeling agent or dye, avoiding the related bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5′-d[(G3T)3G3]-3′ (G3T, the most fluorescent G4 structure reported to date).Fold up
Unconventional Reactivity with DABCO-Bis(sulfur dioxide): C–H Bond Sulfenylation of ImidazopyridinesEuropean Journal of Organic Chemistry : 2020 : 2101-2109 : DOI : 10.1002/ejoc.202000112 Learn more
This work highlights the unexpected and unprecedented outcome of the reactivity with DABCO‐bis(sulfur dioxide). The use of this reagent led to the exclusive introduction of a sulfur atom on the C‐3 position of imidazopyridines instead of a sulfone group. The reaction methodology turned out to be robust, scalable and suitable for various imidazopyridines and aryl iodides both bearing substituents with different electronic and steric properties (38 examples). Beyond the fact that this synthetic method complements the previously reported protocols for sulfenylation reactions, this work is meant to underline the unconventional role of DABCO‐bis(sulfur dioxide).Fold up
Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1Nature Communications : 11 : 1819 : DOI : 10.1038/s41467-020-15689-9 Learn more
The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors.Fold up
Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes.Biochemistry : 59 : 1261-1272 : DOI : 10.1021/acs.biochem.9b01094 Learn more
We investigate herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite that adopt a parallel topology while differing in the length of the central loop (from nine nucleotides to one nucleotide). It is revealed that NCL strongly binds to long-loop (five to nine nucleotides) G4 while interacting weakly with the shorter variants (loop with fewer than three nucleotides). Photo-cross-linking experiments using 5-bromo-2′-deoxyuridine (BrU)-modified sequences further confirmed the loop-length dependency, thereby indicating that the WT-CEB25-L191 (nine-nucleotide loop) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the product(s) obtained by photo-cross-linking NCL to this sequence enabled the identification of one contact site corresponding to a 15-amino acid fragment located in helix α2 of RNA binding domain 2 (RBD2), which sheds light on the role of this structural element in G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL-G4 interaction was explored. It was found that only the most potent ligand PhenDC3 can inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study describes the molecular mechanism by which NCL recognizes G4-containing long loops and leads to the proposal of a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop-quartet interaction.Fold up
A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolutionbioRxiv : DOI : 10.1101/2020.02.26.965269 Learn more
Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.Fold up
Year of publication 2019
Deciphering the mechanism of inhibition of SERCA1a by sarcolipin using molecular simulationsbioRxiv : DOI : 10.1101/2019.12.17.879825 Learn more
SERCA1a is an ATPase calcium pump that transports Ca2+ from the cytoplasm to the sarco/endoplasmic reticulum lumen. Sarcolipin (SLN), a transmembrane peptide, regulates the activity of SERCA1a by decreasing its Ca2+ transport rate, but its mechanism of action is still not well understood. To decipher this mechanism, we have performed normal modes analysis in the all-atom model, with the SERCA1a-SLN complex or the isolated SERCA1a embedded in an explicit membrane. The comparison of the results allowed us to provide an explanation for the action of SLN that is in good agreement with experimental observations. In our analyses, the presence of SLN locally perturbs the TM6 transmembrane helix and as a consequence modifies the position of D800, one of the key metal-chelating residues. Additionally, it reduces the flexibility of the gating residues, V304 and E309 in TM4, at the entrance of the Ca2+ binding sites, which would decrease the affinity for Ca2+. Unexpectedly, SLN has also an effect on the ATP binding site more than 35 r A away, due to the straightening of TM5, a long helix considered as the spine of the protein. The straightening of TM5 modifies the structure of the P-N linker that sits above it, and which comprises the 351DKTG354 conserved motif, resulting in an increase of the distance between ATP and the phosphorylation site. As a consequence, the turn-over rate could be affected. All this gives SERCA1a the propensity to go toward a Ca2+-deprived E2-like state in the presence of SLN and toward a Ca2+ high-affinity E1-like state in the absence of SLN, although the SERCA1a-SLN complex was crystallized in an E1-like state. In addition to a general mechanism of inhibition of SERCA1a regulatory peptides, this study also provides an insight in the conformational transition between the E2 and E1 states.Fold up
DNA junction ligands trigger DNA damage and are synthetic lethal with DNA repair inhibitors in cancer cellsJournal of the American Chemical Society : 142 : 424-435 : DOI : 10.1021/jacs.9b11150 Learn more
Translocation of DNA and RNA polymerases along their duplex substrates results in DNA supercoiling. This torsional stress promotes the formation of plectonemic structures, including three-way DNA junction (TWJ), which can block DNA transactions and lead to DNA damage. While cells have evolved multiple mechanisms to prevent the accumulation of such structures, stabilizing TWJ through ad hoc ligands offer an opportunity to trigger DNA damage in cells with high level of transcription and replication, such as cancer cells. Here, we develop a series of azacryptand-based TWJ ligands, we thoroughly characterize their TWJ-interacting properties in vitro and demonstrate their capacity to trigger DNA damage in rapidly dividing human cancer cells. We also demonstrate that TWJ ligands are amenable to chemically induced synthetic lethality strategies upon association with inhibitors of DNA repair, thus paving the way towards innovative drug combinations to fight cancers.Fold up
The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas.Acta neuropathologica communications : 7 : 175 : DOI : 10.1186/s40478-019-0833-0 Learn more
All cancer cells need to maintain functional telomeres to sustain continuous cell division and proliferation. In human diffuse gliomas, functional telomeres are maintained due either to reactivation of telomerase expression, the main pathway in most cancer types, or to activation of a mechanism called the alternative lengthening of telomeres (ALT). The presence of IDH1/2 mutations (IDH-mutant) together with loss of ATRX expression (ATRX-lost) are frequently associated with ALT in diffuse gliomas. However, detection of ALT, and a fortiori its quantification, are rarely, if ever, measured in neuropathology laboratories. We measured the level of ALT activity using the previously described quantitative “C-circle” assay and analyzed it in a well characterized cohort of 104 IDH-mutant and ATRX-lost adult diffuse gliomas. We report that in IDH-mutant ATRX-lost anaplastic astrocytomas, the intensity of ALT was inversely correlated with age (p < 0.001), the younger the patient, the higher the intensity of ALT. Strikingly, glioblastomas having progressed from anaplastic astrocytomas did not exhibit this correlation. ALT activity level in the tumor did not depend on telomere length in healthy tissue cells from the same patient. In summary, we have uncovered the existence, in anaplastic astrocytomas but not in glioblastomas with the same IDH and ATRX mutations, of a correlation between patient age and the level of activity of ALT, a telomerase-independent pathway of telomere maintenance.Fold up
TGF-β-induced fibrotic stress increases G-quadruplex formation in human fibroblasts.FEBS letters : 593 : 3149-3161 : DOI : 10.1002/1873-3468.13658 Learn more
Scar formation after wound healing is a major medical problem. A better understanding of the dynamic nuclear architecture of the genome during wound healing could provide insights into the underlying pathophysiology and enable novel therapeutic strategies. Here, we demonstrate that TGF-β- induced fibrotic stress increases formation of the dynamic secondary DNA structures called G-quadruplexes in skin fibroblasts, which is coincident with increased expression of collagen 1. This G-quadruplex formation is attenuated by a small molecule inhibitor of intracellular Ca influx and an anti-fibrotic compound. In addition, we identify G-quadruplex-forming sequences in the promoter region of COL1A1, which encodes collagen 1, and confirm their ability to form G-quadruplex structures under physiologically relevant conditions. Our findings reveal a link between G-quadruplexes and scar formation that may lead to novel therapeutic interventions.Fold up