Biology subjects

Ortega, R.

Publications and source records attributed to Ortega, R..

12 recordsLinked to original sources

bioRxiv2025

Synchrotron XRF imaging reveals manganese accumulation in the Golgi and post-synapses of neurons and enhanced uptake in astrocytes

Manganese is an essential trace metal for humans, but excessive exposure can cause neurotoxicity, including parkinsonian syndromes, cognitive deficits, and has also been implicated in the pathogenesis of neurodegenerative diseases. Globally, tens of millions of people are exposed to elevated manganese levels through drinking water, exceeding the WHO recommended guideline. Despite its public health significance, the cellular and subcellular mechanisms underlying manganese neurotoxicity remain poorly defined, particularly its distribution across brain cell types and its specific intracellular targets. In this study, we investigated manganese accumulation in primary cultures of hippocampal neurons and astrocytes. Using a correlative imaging approach that combined cryo-fluorescence light microscopy with synchrotron X-ray fluorescence imaging, we mapped and quantified manganese at subcellular resolution. Our analysis revealed that manganese preferentially accumulates in the Golgi apparatus of both neurons and astrocytes. In neurons, manganese was also detected in dendrites at the postsynaptic density, suggesting a role in synaptic vulnerability. Quantitative elemental analysis showed that astrocytes accumulated 3 times more manganese than neurons. Furthermore, when neurons were co-cultured with astrocytes, their manganese uptake was significantly reduced, indicating a possible protective or buffering role of astrocytes. These findings identify key cellular and subcellular targets of manganese and highlight the Golgi apparatus as a major regulator of manganese neurotoxicity. This work provides a foundation for understanding cell type-specific responses to manganese exposure and may inform the development of targeted neuroprotection strategies. Significance statementManganese neurotoxicity impact millions of people worldwide. Understanding how manganese distributes within brain cells is critical for addressing its neurotoxic effects. This study identifies the Golgi apparatus and postsynaptic density as key sites of manganese accumulation and reveals astrocytes as major regulators of neuronal manganese uptake. These findings provide a cellular framework for developing targeted strategies to mitigate manganese-induced neurotoxicity.

neuroscience↗

bioRxiv2025

PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining during mitosis.

Poly ADP-ribose polymerase (PARP) inhibitors are standard of care treatment for cancers with homologous-recombination deficiencies. Yet, as tumors develop resistance, complementary strategies are emerging, including targeting microhomology-mediated end joining (MMEJ). Given that PARP1 is widely described as a key promoter of MMEJ, one would expect PARP inhibition to suppress MMEJ--potentially making MMEJ inhibition redundant. MMEJ was originally described as a backup pathway, with seminal work linking PARP1 to MMEJ conducted in NHEJ-deficient cells, where MMEJ can be reactivated in G1. However, we now appreciate that MMEJ is a mitotic repair mechanism, but the function of PARP1 in this context remains unclear. Here, we systematically explore the effect that PARP/PARPi have on MMEJ activity in cells with intact NHEJ. Surprisingly, PARP inhibition leads to elevated Pol{theta}-dependent MMEJ levels at ISceI-mediated DSBs, which we find is due to PARP1s regulation of competing pathways at these breaks. Importantly, we show that PARP is dispensable for MMEJ at double-ended DSBs and is expendable for repair of DSBs during mitosis. Altogether, this data shifts the understanding of PARPs role in MMEJ and DNA repair pathway choice. Moreover, we believe this data further strengthens a rationale for PARPi/MMEJi combinatorial drug treatment in HR-deficient cancers.

molecular biology↗

bioRxiv2024

Storage and transport of labile iron is mediated by lysosomes in axons and dendrites of hippocampal neurons

Iron dyshomeostasis in neurons, involving iron accumulation and abnormal redox balance, is implicated in neurodegeneration. In particular, labile iron, a highly reactive pool of intracellular iron, plays a prominent role in iron-induced neurological damage. However, the mechanisms governing the detoxification and transport of labile iron within neurons are not fully understood. This study investigates the storage and transport of labile ferrous iron Fe(II) in cultured primary rat hippocampal neurons. Iron distribution was studied using live cell confocal microscopy with a selective labile Fe(II) fluorescent dye, and synchrotron X-ray fluorescence microscopy (SXRF) for total iron distribution. Fluorescent labelling of the axon initial segment and of lysosomes allowed iron distribution to be correlated with these subcellular compartments. The results show that labile Fe(II) is stored in lysosomes within somas, axons and dendrites and that lysosomal labile Fe(II) is transported retrogradely and anterogradely along axons and dendrites. In addition, we have developed a methodological workflow to quantify labile Fe(II) relative to total iron in neurites. This method is based on correlative imaging of fluorescence microscopy of labile Fe(II) combined with quantitative elemental mapping of total iron by SXRF. Quantitative analysis revealed that after Fe(II) exposure, lysosomal Fe(II) accounts for a small but significant percentage of the total iron content in neurites. These result suggest that after exposure to labile Fe(II), iron is mainly present in a non-reactive form in neurons, while the smaller fraction of reactive labile Fe(II) is stored in lysosomes and can be transported along dendrites and axons.

neuroscience↗

bioRxiv2024

Impaired maturation of wild-type superoxide dismutase 1 associated with neurodegeneration in Parkinson disease brain and a novel murine model

Atypical wild-type superoxide dismutase 1 (SOD1) protein misfolding and deposition occurs specifically within the degenerating substantia nigra pars compacta (SNc) in Parkinson disease. Mechanisms driving the formation of this pathology and relationship with SNc dopamine neuron health, are yet to be fully understood. We applied proteomic mass spectrometry and synchrotron-based biometal quantification to post-mortem brain tissues from the SNc of Parkinson disease patients and age-matched controls to uncover key factors underlying the formation of wild-type SOD1 pathology in this disorder. We then engineered brain copper deficiency and upregulated SOD1 protein levels in a novel mouse strain, termed the SOCK mouse, to verify their involvement in the development of Parkinson-like wild-type SOD1 pathology and their impact on dopamine neuron health. Soluble SOD1 protein in the degenerating Parkinson disease SNc exhibited altered post-translational modifications, which may underlie changes to the enzymatic activity and aggregation of the protein in this region. These include decreased copper binding, dysregulation of physiological glycosylation, and atypical oxidation and glycation of key SOD1 amino acid residues. We demonstrated that the biochemical profile introduced in SOCK mice promotes the same post-translational modifications and the development of Parkinson-like wild-type SOD1 pathology in the midbrain and cortex. This pathology accumulates progressively with age and is accompanied by nigrostriatal degeneration and dysfunction, which occur in the absence of -synuclein deposition. These mice do not exhibit weight loss nor spinal cord motor neuron degeneration, distinguishing them from transgenic mutant SOD1 mouse models. This study provides the first in vivo evidence that mismetallation and altered post-translational modifications precipitates wild-type SOD1 misfolding, dysfunction and deposition in the Parkinson disease brain, which may contribute to SNc dopamine neuron degeneration. Our data position this pathology as a novel drug target for this disorder, with a particular focus on therapies capable of correcting alterations to SOD1 post-translational modifications.

neuroscience↗

bioRxiv2023

Fate of telomere entanglements is dictated by the timing of anaphase midregion nuclear envelope breakdown

Persisting replication intermediates can confer mitotic catastrophe if left unresolved. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric replication fork stalling and in turn, telomere entanglements that stretch between the segregating chromosomes at anaphase. At [≤]20{degrees}C, these entanglements fail to resolve, resulting in lethality. Rif1, a conserved DNA replication/repair protein, localizes between the segregating chromosomes and specifically hinders the resolution of telomere entanglements without affecting their formation. During anaphase, the spindle and the last segments of segregating chromatin are encased by the nuclear envelope (NE), creating a microdomain termed the anaphase midregion. In the final stages of fission yeast mitosis, this midregion undergoes local NE breakdown. Here we demonstrate that in response to taz1{Delta} telomeric entanglements, Rif1 delays midregion NE breakdown, and this delay disfavors entanglement resolution. Accordingly, gene deletions that hasten midregion NE breakdown phenocopy, and are epistatic with, rif1+ deletion. Conversely, gene deletions that delay midregion NE breakdown block the taz1{Delta} telomere detanglement afforded by loss of Rif1. Overexpression of Rif1 in a wild type background causes cold-specific NE defects and lethality, which are rescued by treatment with a membrane fluidizing agent. We propose that delayed NE breakdown normally favors the resolution of simple entanglements, arising from incomplete replication, by delaying exposure to the cytoplasm. In contrast, resolution of more complex entanglements involving strand invasion, like entanglements between nonsister taz1{Delta} telomeres, requires more rapid exposure to the cytoplasm. These observations uncover an unexpected coordination between NE remodeling and DNA processing events that can prevent or promote aneuploidy.

molecular biology↗

bioRxiv2023

Combinatory EHMT and PARP inhibition induces an interferon response and a CD8 T cell-dependent tumor regression in PARP inhibitor-resistant models

Euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/2), which catalyze demethylation of histone H3 lysine 9 (H3K9me2), contribute to tumorigenesis and therapy resistance through unknown mechanisms of action. In ovarian cancer, EHMT1/2 and H3K9me2 are directly linked to acquired resistance to poly-ADP-ribose polymerase (PARP) inhibitors and are correlated with poor clinical outcomes. Using a combination of experimental and bioinformatic analyses in several PARP inhibitor resistant ovarian cancer models, we demonstrate that combinatory inhibition of EHMT and PARP is effective in treating PARP inhibitor resistant ovarian cancers. Our in vitro studies show that combinatory therapy reactivates transposable elements, increases immunostimulatory dsRNA formation, and elicits several immune signaling pathways. Our in vivo studies show that both single inhibition of EHMT and combinatory inhibition of EHMT and PARP reduces tumor burden, and that this reduction is dependent on CD8 T cells. Together, our results uncover a direct mechanism by which EHMT inhibition helps to overcome PARP inhibitor resistance and shows how an epigenetic therapy can be used to enhance anti-tumor immunity and address therapy resistance.

cancer biology↗

bioRxiv2022

The APE2 nuclease is essential for DNA double strand break repairby microhomology-mediated end-joining.

Microhomology-mediated end-joining (MMEJ) is an intrinsically mutagenic pathway of DNA double strand break repair essential for proliferation of homologous recombination (HR) deficient tumors. While targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as a novel MMEJ effector. We show that loss of APE2 blocks the fusion of deprotected telomeres by MMEJ and inhibits MMEJ in DNA repair reporter assays to levels comparable to Pol Theta suppression. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease domain and further identify uncharacterized domains required for recruitment to damaged DNA. We conclude that HR-deficient cells are addicted to APE2 due to a previously unappreciated role in MMEJ, which could be exploited in the treatment of cancer.

molecular biology↗

bioRxiv2021

BATL: Bayesian annotations for targeted lipidomics

MotivationBioinformatic tools capable of annotating, rapidly and reproducibly, large, targeted lipidomic datasets are limited. Specifically, few programs enable high-throughput peak assessment of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) data acquired in either selected or multiple reaction monitoring (SRM and MRM) modes. ResultsWe present here Bayesian Annotations for Targeted Lipidomics (BATL), a Gaussian naive Bayes classifier for targeted lipidomics that annotates peak identities according to eight features related to retention time, intensity, and peak shape. Lipid identification is achieved by modelling distributions of these eight input features across biological conditions and maximizing the joint posterior probabilities of all peak identities at a given transition. When applied to sphingolipid and glycerophosphocholine SRM datasets, we demonstrate over 95% of all peaks are rapidly and correctly identified. Availability and implementationBATL software is freely accessible online at https://complimet.ca/batl/ and is compatible with Safari, Firefox, Chrome and Edge. Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2020

The SINGLE FLOWER (SFL) gene encodes a MYB transcription factor that regulates the number of flowers produced by the inflorescence of chickpea.

research conducted & rationaleLegume species usually have compound inflorescences, where flowers appear in secondary inflorescences (I2), at lateral positions of the primary inflorescence (I1), in contrast to simple inflorescences, as in Arabidopsis, where flowers are formed in the primary inflorescence stem. The number of flowers per I2, characteristic of each legume species, determines inflorescence diversity, and the number of pods produced, which can affect yield. Gene Regulatory Network that controls the activity of I2 meristems, and therefore the number of flowers per secondary inflorescence is mostly unknown, as well as how specific are factors controlling this trait and whether they share this function in other meristems. methodsChickpea produces one flower per I2 but single flower (sfl) mutants produce two (double-pod phenotype). By mapping the sfl-d mutation and identification and analysis of a second mutant allele we have isolated SFL. We used scanning electron microscopy to study the effect of sfl mutations on inflorescence ontogeny and in situ hybridization to study the expression of SFL and of meristem identity genes in the developing chickpea inflorescence. key resultWe show that the SFL gene corresponds to CaRAX1/2a, encoding a MYB transcription factor. Our results show that CaRAX1/2a / SFL is specifically expressed in the I2 meristem, possibly activated by CaVEGETATIVE1. main conclusion & key points for discussionOur findings reveal that SFL plays a central role in the control of chickpea inflorescence architecture, specifically acting in the I2 meristem to control the time length for which it is active, and therefore determining the number of floral meristems that it can produce.

plant biology↗

bioRxiv2020

Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations

Uranium exposure can lead to neurobehavioral alterations in particular of the monoaminergic system, even at non-cytotoxic concentrations. However, the mechanisms of uranium neurotoxicity after non-cytotoxic exposure are still poorly understood. In particular, imaging uranium in neurons at low intracellular concentration is still very challenging. We investigated uranium intracellular localization by means of synchrotron X-ray fluorescence imaging with high spatial resolution (< 300 nm) and high analytical sensitivity (< 1 g.g-1 per 300 nm pixel). Neuron-like SH-SY5Y human cells differentiated into a dopaminergic phenotype were continuously exposed, for seven days, to a non-cytotoxic concentration (10 M) of soluble natural uranyl. Cytoplasmic submicron uranium aggregates were observed accounting on average for 62% of the intracellular uranium content. In some aggregates, uranium and iron were co-localized suggesting common metabolic pathways between uranium and iron storage. Uranium aggregates contained no calcium or phosphorous indicating that detoxification mechanisms in neuron-like cells are different from those described in bone or kidney cells. Uranium intracellular distribution was compared to fluorescently labeled organelles (lysosomes, early and late endosomes) and to fetuin-A, a high affinity uranium-binding protein. A strict correlation could not be evidenced between uranium and the labelled organelles, or with vesicles containing fetuin-A. Our results indicate a new mechanism of uranium cytoplasmic aggregation after non-cytotoxic uranyl exposure that could be involved in neuronal defense through uranium sequestration into less reactive species. The remaining soluble fraction of uranium would be responsible for protein binding and the resulting neurotoxic effects.

pharmacology and toxicology↗

bioRxiv2020

Estimating daily intakes of manganese due to breast milk, infant formulas, or young child nutritional beverages in the United States and France: Comparison to sufficiency and toxicity thresholds

Background Although manganese (Mn) is an essential nutrient, recent research has revealed that excess Mn in early childhood may have adverse effects on neurodevelopment.Methods We estimated daily total Mn intake due to breast milk at average body weights by reviewing reported concentrations of breast milk Mn and measurements of body weight and breast milk intake at 3 weeks, 4.25 months, 7 months, and 18 months. We compared these figures to the Mn content measured in 44 infant, follow-up, and toddler formulas purchased in the United States and France. We calculated Mn content of formula products made with ultra-trace elemental analysis grade water (0 µg Mn/L) and with water containing 250 µg Mn/L, a concentration which is relatively high but less than the World Health Organization Health-based value of 400 µg Mn/L or the United States Environmental Protection Agency Health Advisory of 350 µg Mn/L.Results Estimated mean daily Mn intake from breast milk ranged from 1.2 µg Mn/kg/day (3 weeks) to 0.16 µg Mn/kg/day (18 months), with the highest intakes at the youngest age stage we considered, 3 weeks. Estimated daily Mn intake from formula products reconstituted with 0 µg Mn/L water ranged from 130 µg Mn/kg/day (3 weeks) to 4.8 µg Mn/kg/day (18 months) with the highest intakes at 3 weeks. Formula products provided 28 to 520 times greater than the mean daily intake of Mn from breast milk for the 4 age stages that we considered. Estimated daily Mn intake from formula products reconstituted with water containing 250 µg Mn/L ranged from 12 µg Mn/kg/day to 170 µg Mn/kg/day, which exceeds the United States Environmental Protection Agency Reference Dose of 140 µg Mn/kg/day for adults.Conclusions Mn deficiency is highly unlikely with exclusive breast milk or infant formula feeding, but established tolerable daily intake levels for Mn may be surpassed by some of these products when following labeled instructions.HighlightsMn deficiency is unlikely with exclusive breast milk or infant formula feeding.Breast milk Mn mean intake is 1.2 µg/kg/day (3 weeks)-0.16 µg/kg/day (18 months).Formula Mn intake range is 130 µg/kg/day (3 weeks)-4.8 µg/kg/day (18 months).Formula products reconstituted with 250 µg Mn/L water may exceed 140 µg Mn/kg/day.Formula products may surpass regulatory tolerable daily intake levels for Mn.Competing Interest StatementThe authors have declared no competing interest.1 List of abbreviationsAIAdequate IntakeANSESAgence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (French Agency for Food, Environmental and Occupational Health & Safety)DJAdose journalière admissible (daily admissible dose, tolerable daily intake)EFSAEuropean Food Safety AuthorityEUEuropean UnionFAOFood and Agriculture OrganizationHBVHealth-Based ValueINSPQInstitut national de santé publique du Québec (National Institute of Public Health of Québec)IOMInstitute of MedicineIQintelligence quotientJECFAJoint FAO/WHO Expert Committee on Food AdditivesLOAELLowest Observed Adverse Effect LevelMnManganeseMnSODmanganese superoxide dismutaseMRmagnetic resonanceNOAELNo Observed Adverse Effect LevelPIXEparticle induced X-ray emissionPNparental nutritionRBSRutherford backscatteringRfDReference DoseSCFScientific Committee on FoodT1RT1 relaxationTPNtotal parenteral nutritionU.S.United States Environmental Protection AgencyEPAEnvironmental Protection AgencyUSUnited StatesView Full Text

pharmacology and toxicology↗

bioRxiv2019

Correlating STED and synchrotron XRF nano-imaging unveils the co-segregation of metals and cytoskeleton proteins in dendrites

Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecular mechanisms behind these processes are still elusive due in part to the difficulty of imaging trace metals together with proteins at the synaptic level. We correlate stimulated emission depletion (STED) microscopy of proteins and synchrotron X-ray fluorescence (SXRF) imaging of trace metals, both performed with 40 nm spatial resolution, on primary rat hippocampal neurons. We achieve a detection limit for zinc of 14 zeptogram (10-21 g) per pixel. We reveal the co-localization at the nanoscale of zinc and tubulin in dendrites with a molecular ratio of about one zinc atom per tubulin-{beta} dimer. We observe the co-segregation of copper and F-actin within the nano-architecture of dendritic protrusions. In addition, zinc chelation causes a decrease in the expression of cytoskeleton proteins in dendrites and spines. Overall, these results indicate new functions for zinc and copper in the modulation of the cytoskeleton morphology in dendrites, a mechanism associated to neuronal plasticity and memory formation.

neuroscience↗