Biology subjects

Hirtz, C.

Publications and source records attributed to Hirtz, C..

14 recordsLinked to original sources

bioRxiv2026

SPEAR: Spatial Epitranscriptomic Analysis by Mass Spectrometry Reveals Localized RNA Modification Remodeling During Glioma Progression

RNA modifications shape cell identity and disease, yet their spatial organization within tissues has remained largely inaccessible to existing technologies. NGS-based methods can map a limited number of individual modifications with spatial resolution, while liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides comprehensive and quantitative coverage of the epitranscriptome from bulk, homogenized tissue. No current approach combines histological resolution, broad modification coverage, quantitative accuracy, and the sensitivity required for microscale clinical specimens. We present SPEAR (SPatial Epitranscriptomic Analysis by mass spectrometRy), a workflow coupling laser microdissection with a redesigned LC-MS/MS pipeline to enable quantitative epitranscriptomic profiling directly from formalin-fixed, paraffin-embedded (FFPE) tissue. This combination enables reproducible detection of dozens of RNA modifications from regions as small as 0.1 mm2, establishing a spatial scale previously inaccessible to mass spectrometry-based approaches. Applied to serial mouse brain sections, SPEAR resolved anatomically defined epitranscriptomic signatures across white and gray matter, establishing its spatial resolution and reproducibility in a controlled system. We then applied SPEAR to human diffuse gliomas containing, within individual specimens, both primary low-grade tumor regions and discrete foci undergoing transformation toward higher-grade malignancy. SPEAR resolved this transition directly, revealing a coordinated decrease in multiple methylated RNA modifications specifically within higher-grade regions, a spatially restricted signal that was masked by bulk analysis and reproducibly observed across patients despite underlying genetic heterogeneity. SPEAR establishes spatial epitranscriptomics as a tractable approach for molecular profiling of clinical tissue, providing a platform for biomarker discovery and molecular stratification based on the spatial organization of RNA modifications.

cancer biology↗

bioRxiv2025

Reversible multiscale corneal sexual dimorphism orchestrates neuro-epithelial coupling and tear proteome composition

Donor-recipient sex mismatch is an under-recognized risk factor for corneal graft rejection, yet the biological underpinnings at the ocular surface remain undefined. We hypothesized that sex hormones coordinately tune the tear-nerve-epithelium axis and that androgen exposure could normalize sex-linked differences relevant to alloimmune risk. We profiled 12-week-old mice in three hormonal contexts, i.e. male, female, and testosterone-treated female, combining tear biochemistry and label-free proteomics with epithelial lineage dynamics, in vivo sensory function, and corneal/trigeminal transcriptional readouts. Tear collection rate was comparable across groups, but total tear protein was reduced in females. Proteomics revealed extensive sex differences in extracellular composition, spanning lipid transport, protease-antiprotease balance, complement activity, and secretory/mucin pathways. Epithelial analyses showed sex-linked differences in progenitor output and spatial deployment, and sensory metrics indicated divergent innervation architecture and function. Across modalities, androgen supplementation in females shifted molecular and physiological profiles toward the male state, attenuating nearly all dimorphic signals, demonstrating that close to all sexual dimorphism observed here is reversible by testosterone and reflects an actively maintained endocrine state rather than a fixed developmental program. Complementary metabolite profiling provides a sex-stratified atlas of free modified nucleosides in tears, positioning extracellular epitranscriptomic markers as accessible indicators of hormonal context. Together, these results establish a multiscale, hormone-responsive sexual dimorphism at the ocular surface and offer a mechanistic framework linking sex and endocrine status to parameters that influence graft integration. They motivate sex-aware biomarkers, stratification by hormonal context in diagnostics and trials, and therapeutic strategies, including androgenic modulation, to mitigate the elevated rejection risk associated with sex mismatch in corneal transplantation.

physiology↗

bioRxiv2025

Loss of tRNA uridine thiolation affects mRNA translation, protein production, and sulfur-compound metabolism in Arabidopsis

The uridine at position 34 of tRNA anticodon loops is always modified at variable levels depending on environmental conditions, but a function for this highly conserved modification has not been firmly established. Using Arabidopsis thaliana, we show that the thiolation of U34 is a prerequisite for the subsequent modifications at position 32 and 37 of the tRNALys(UUU) anticodon loop, revealing a novel modification network. Surprisingly, the level of tRNALys(UUU) is strongly increased rather than reduced in the ctu1 or ctu2 mutant backgrounds that prevent these modifications. This suggests the existence of a regulatory feedback loop that drives the transcription of this specific tRNA gene family. Furthermore, we observed that the ability of the ribosome to decode AAA, GAA and CAA codons is impaired when the thiol group is lost, leading to a reduction in protein production, especially for genes enriched in these codons. Finally, we show that loss of tRNA thiolation results in variations in levels of many proteins involved in sulfur-compound metabolism and several sulfur-containing metabolites, suggesting that the level of tRNA thiolation may act as a sensor that regulates these processes.

molecular biology↗

bioRxiv2025

RABV L protein plays a role in immune escape through its methyltransferase activity

Viruses in the Mononegavirales order encode a large protein that orchestrates replication, transcription, and the capping of viral RNA. This protein, comprising over 2.000 amino acids, contains an RNA-dependent RNA polymerase, a capping domain, and a methyltransferase (MTase) domain involved in methylating the cap structure. The MTase domain features a conserved K-D-K-E catalytic tetrad -typical of 2'O-methyltransferases-which is essential for methylating viral mRNA caps at both the N7 and 2'O positions. However, the role of these residues in other epitranscriptomic modifications of rabies virus (RABV) RNAs remains poorly characterized. To further explore the role of mRNA cap methylation in the immune evasion strategies of RABV, we investigated the functional contribution of the K-D-K-E motif within the MTase domain, using the Thai isolate as a model. Using reverse genetics, we demonstrated that the mutation K1830R in the K-D-K-E tetrad of the Tha MTase domain induces changes in the methylation landscape of viral mRNAs and, intriguingly, of host mRNAs. In addition, viruses harbouring the K1830R mutation are more sensitive to interferon- and exhibit a less pathogenic phenotype in vitro and in vivo compared to the wild-type virus. Overall, these results suggest that the regulation of viral and cellular RNA methylation landscapes plays a crucial role in controlling RABV infection. Although the exact role of these epitranscriptomic modifications is not yet fully understood, some of these methylations appear to have proviral effects and enhance viral propagation by allowing RABV to efficiently evade the hosts antiviral response. ImportanceThis study highlights the pivotal role of the K-D-K-E catalytic domain included in the methyltransferase domain of the large protein of Rabies virus, by modelling viral RNAs with epitranscriptomic changes. For the first time, we identify specific methylations on the viral RNA, such as 2-O and m6A methylations, which seem to enable the virus to mask its RNA and evade detection by the hosts pattern recognition receptors. These epitranscriptomic modifications affect not only viral RNAs but also cellular RNAs, underscoring a complex interplay between viral and host mechanisms. We further demonstrate that RABV harbouring an altered K-D-R-E catalytic domain, exhibit differential methylation patterns correlated with increased sensitivity to IFN and lower pathogenicity. This emphasizes the importance of this domain in virulence and immune evasion.

microbiology↗

bioRxiv2025

Integrated tear proteomics define the molecular blueprint of corneal epithelial repair

The tear film plays an essential role in corneal protection and regeneration following injury. Although the cornea is a structurally conserved organ across terrestrial vertebrates, the extent to which tear film mediated wound healing responses are evolutionarily conserved remains unclear. This study aimed to identify core and species-specific molecular pathways activated in the tear film during corneal wound healing in humans and mice. We conducted a meta analysis of tear proteomic datasets from human subjects undergoing photorefractive keratectomy (PRK) and mice subjected to mechanical corneal abrasion. Differentially expressed proteins were identified and subjected to Reactome and Gene Ontology (GO) enrichment analyses to determine conserved and divergent biological responses. Approximately one third of the tear film proteomic response to corneal injury was conserved across species. Shared upregulated pathways included complement activation, actin cytoskeletal remodeling, protein synthesis, and acute inflammatory responses. Simultaneously, pathways related to adaptive immunity, proteolysis, and general metabolism were consistently downregulated. Human specific responses were enriched in secretory pathways, vesicle trafficking, and immune surveillance, whereas murine specific responses highlighted mitochondrial activation, oxidative metabolism, and stress adaptation. These distinctions reflect species dependent physiological strategies in managing epithelial repair. Our findings reveal a conserved molecular framework that governs corneal wound healing across species, with notable species specific adaptations. This cross species comparison underscores the translational relevance of tear film analysis and supports the development of targeted therapies tailored to human specific wound healing mechanisms in ocular surface disease.

physiology↗

bioRxiv2025

Temporal analysis of tear fluid proteome reveals critical corneal repair events after photorefractive surgery

Corneal epithelial wound healing is a complex and finely orchestrated process critical for restoring visual acuity following injury or surgery. Photorefractive keratectomy (PRK), a common refractive surgical procedure, provides an ideal clinical model to study this process. Here, we employed advanced proteomic analysis to comprehensively map the dynamic changes in the tear fluid proteome at distinct phases following PRK. Our findings revealed significant alterations in nearly 45% of the tear proteome, highlighting temporally distinct molecular signatures. Immediately post-injury, a robust but controlled anti-inflammatory response coincided with pronounced upregulation of protein synthesis and cellular stress-management pathways. Subsequently, at day three, the molecular landscape shifted toward sustained epithelial regeneration, extracellular matrix remodeling, and controlled inflammation resolution. By delineating these critical and temporally compartmentalized molecular events, this study identifies novel tear-based biomarkers indicative of corneal healing efficacy and provides essential insights into potential therapeutic targets for improving clinical outcomes in corneal wound healing and ocular surface disorders.

physiology↗

bioRxiv2025

Decoding the molecular complexity governing corneal wound closure in vivo

The cornea, the transparent outermost layer of the eye, possesses exceptional wound healing capabilities essential for vision preservation. The complexity of the corneal microenvironment is central to its rapid healing; however, the molecular mechanisms orchestrating this process remain poorly defined, limiting therapeutic advancements. Here, we elucidate the extensive remodeling of the corneal molecular landscape following physical injury. Multi-omics analyses--including transcriptomic, epitranscriptomic, and proteomic profiling--uncover significant induction of epithelial cell plasticity driving wound closure. Moreover, lacrimal gland ablation further suppresses Pax6 expression, highlighting its regulatory role. Our multi-omic approach uniquely reveals bilateral remodeling of the molecular environment, a phenomenon constrained by an intact tear film. Collectively, our findings identify novel molecular factors critical to corneal healing, significantly advancing the understanding of epithelial plasticity. These insights will facilitate the translation of cell plasticity research into innovative strategies for tissue and organ regeneration.

physiology↗

bioRxiv2024

Dual CRALBP isoforms unveiled: iPSC-derived retinal modelling and AAV2/5-RLBP1 gene transfer raise considerations for effective therapy

Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous disorders characterised by progressive vision loss. Over 270 causative genes have been identified and variants within the same gene can give rise to clinically distinct disorders. Human induced pluripotent stem cells (iPSCs) have revolutionised disease modelling, by allowing pathophysiological and therapeutic studies in the patient and tissue context. The IRD gene RLBP1 encodes CRALBP, an actor of the rod and cone visual cycles in the retinal pigment epithelium (RPE) and Muller cells, respectively. Variants in RLBP1 lead to three clinical subtypes: Bothnia dystrophy, Retinitis punctata albescens and Newfoundland rod-cone dystrophy. We modelled RLBP1-IRD subtypes by patient-specific iPSC-derived RPE and identified pertinent therapeutic read-outs. We developed an AAV2/5-mediated gene replacement strategy and provided a proof-of-concept in the ex vivo human models that was validated in an in vivo Rlbp1-/- murine model. Most importantly, we identified a previously unsuspected smaller CRALBP isoform that is naturally and differentially expressed in both human and murine retina. The new isoform arises from an alternative methionine initiation site and plays a role in the visual cycle. This work provides novel insights into CRALBP expression and RLBP1-associated pathophysiology and raises important considerations for successful gene supplementation therapy.

molecular biology↗

bioRxiv2024

Inherited mitochondrial dysfunction triggered by OPA1 mutation impacts the sensory innervation fibre identity, functionality and regenerative potential in the cornea

Mitochondrial dysfunctions are detrimental to organ metabolism. The cornea, transparent outmost layer of the eye, is prone to environmental aggressions, such as UV light, and therefore dependent on adequate mitochondrial function. While several reports have linked corneal defects to mitochondrial dysfunction, the impact of OPA1 mutation, known to induce such dysfunction, has never been studied in this context. We used the mouse line carrying OPA1delTTAG mutation to investigate its impact on corneal biology. To our surprise, neither the tear film composition nor the corneal epithelial transcriptomic signature were altered upon OPA1 mutation. However, when analyzing the corneal innervation, we discovered an undersensitivity of the cornea upon the mutation, but an increased innervation volume at 3 months. Furthermore, the fibre identity changed with a decrease of the SP+ axons. Finally, we demonstrated that the innervation regeneration was less efficient and less functional in OPA1+/- corneas. Altogether, our study describes the resilience of the corneal epithelial biology, reflecting the mitohormesis induced by the OPA1 mutation, and the adaptation of the corneal innervation to maintain its functionality despite its morphogenesis defects. These findings will participate to a better understanding of the mitochondrial dysfunction on peripheral innervation.

pathology↗

bioRxiv2024

A candidate reference method for the quantification of α-synuclein in cerebrospinal fluid using an SI traceable primary calibrator and multiple reaction monitoring

Objectives-synuclein aggregation is an indicator of neurodegenerative diseases such as Parkinsons disease (PD) and recent advances have suggested that this protein could serve as a potential biomarker. It has been indicated that soluble and oligomeric -synuclein in biological fluids could have diagnostic applications for PD. Clinical laboratories currently rely on antibody-based assays to detect -synuclein. These assays have limited specificity, low sensitivity and poor inter-lab reproducibility, which prevents the validation of -synuclein as a biomarkers. This study aims to fill the unmet need for the standardisation of clinical measurements for -synuclein. MethodsWe report the first candidate reference method for -synuclein, using an SI traceable primary calibrator for -synuclein and isotope dilution mass spectrometry. The primary calibrator was traceably quantified utilising a combination of amino acid analysis and nuclear magnetic resonance. A targeted sample clean-up procedure involving a non-denaturing Lys-C digestion and solid-phase extraction allowed for the sensitive detection of multiple proteotypic -synuclein peptides in cerebrospinal fluid (CSF) samples. ResultsThe candidate reference method procedure showed linearity across three orders of magnitude, covering the physiological levels of -synuclein in CSF (LOQ = 0.1 ng/g). The method was used to quantify a cohort of CSF samples and the measurements were correlated with immunoassay-based quantifications. ConclusionsThe SI traceable quantification of -synuclein in complex biological matrices means that the role of this protein can be further elucidated in synucleinopathies. This candidate reference method would lead to the harmonisation of -synuclein measurements, which may allow for development of high throughput clinical tests.

biochemistry↗

bioRxiv2023

Modeling the simultaneous dynamics of proteins in blood plasma and the cerebrospinal fluid in human in vivo

The analysis of protein dynamics or turnover in patients has the potential to reveal altered protein recycling such as in Alzheimer disease, and to provide informative data regarding drug efficacy, or certain biological processes. The observed protein dynamics in a solid tissue or a fluid is the net result of protein synthesis and degradation, but also transport across biological compartments. We report an accurate 3-biological compartment model able simultaneously account for the protein dynamics observed in blood plasma and the cerebrospinal fluid (CSF) including a hidden central nervous system (CNS) compartment. We successfully applied this model to 69 proteins of a single individual displaying similar or very different dynamics in plasma and CSF. This study put a strong emphasis on the methods and tools needed develop this type of model. We believe it will be useful to any researcher dealing with protein dynamics data modeling.

systems biology↗

bioRxiv2023

Enabling population protein dynamics through Bayesian modeling

The knowledge of protein dynamics or turnover in patients provides invaluable information related to certain diseases, drug efficacy, or biological processes. A great corpus of experimental and computational methods has been developed, including by us, in the case of human patients followed in vivo. Moving one step further, we propose here a new modeling approach to capture the highly relevant notion of population protein dynamics. Using two data sets, we show that models inspired by population pharmacokinetics can accurately capture protein turnover within a cohort of individuals, even in presence of substantial inter-individual variability. Such models pave the way for comparative studies searching for altered dynamics or biomarkers in diseases.

systems biology↗

bioRxiv2022

E4F1 COORDINATES PYRUVATE METABOLISM AND THE ACTIVITY OF THE ELONGATOR COMPLEX TO ENSURE PROTEIN TRANSLATION FIDELITY DURING NEURONAL DEVELOPMENT

Pyruvate metabolism defects lead to severe neuropathies such as the Leigh syndrome (LS) but the molecular mechanisms underlying neuronal cell death remain poorly understood. Here, we unravel a connection between pyruvate metabolism and the regulation of the epitranscriptome that is relevant to LS pathogenesis. We identified the transcription factor E4F1 as a key coordinator of AcetylCoenzyme A (AcCoA) production by the pyruvate dehydrogenase complex (PDC) and its utilization as an essential co-factor by the Elongator complex to acetylate tRNAs at the wobble position uridine 34 (U34). E4F1-mediated direct transcriptional regulation of Dlat and Elp3, two genes encoding key subunits of the PDC and of the Elongator complex, respectively, ensured proper translation fidelity and cell survival in the central nervous system (CNS) during mouse embryonic development. Furthermore, analysis of PDH-deficient cells highlighted a crosstalk linking the PDC to ELP3 expression that is perturbed in LS patients.

developmental biology↗

bioRxiv2019

In Vivo Large Scale Mapping Of Protein Turnover In The Human Cerebrospinal Fluid

The extraction of accurate physiological parameters from clinical samples provides a unique perspective to understand disease etiology and evolution, including under therapy. We introduce a new proteomics framework to map patient proteome dynamics in vivo, either proteome wide or in large targeted panels. We applied it to ventricular cerebrospinal fluid (CSF) and could determine the turnover parameters of almost 200 proteins, whereas a handful were known previously. We covered a large number of neuron biology- and immune system-related proteins including many biomarkers and drug targets. This first large data set unraveled a significant relationship between turnover and protein origin that relates to our ability to investigate the central nervous system physiology precisely in future studies. Our data constitute a reference in CSF biology as well as a repertoire of peptides for the community to design new proteome dynamics analyses. The disclosed methods apply to other fluids or tissues provided sequential sample collection can be performed.

systems biology↗