Biology subjects

Ghesquiere, B.

Publications and source records attributed to Ghesquiere, B..

16 recordsLinked to original sources

bioRxiv2026

COG5-mediated Golgi organization coordinates neural progenitor fate during human brain development

Apical radial glial cells (aRGs) are highly polarized epithelial cells that drive human cortical development through tightly regulated lineage progression. A distinctive feature of aRGs is their non-canonical Golgi organization, raising the possibility that specialized intracellular trafficking and secretory processes contribute to progenitor behaviour. However, how Golgi architecture contributes to human corticogenesis remains poorly understood. Here, we investigate this question in the context of COG5 deficiency, a rare congenital disorder of glycosylation associated with neurodevelopmental defects, using patient-derived fibroblasts and brain organoids, N-glycomics, glycolipidomics, and single-cell transcriptomics. We show that COG5 deficiency disrupts Golgi organization and impairs Golgi-to-ER retrograde trafficking. At the neurodevelopmental level, COG5-deficient organoids display altered ventricular organization and reduced basal progenitor abundance. Single-cell transcriptomic analysis reveals rewired developmental trajectories characterized by premature differentiation from apical progenitors with activation of neuronal pathways. Despite the absence of major abnormalities in plasma glycoproteins glycosylation, glycolipidomic analysis identified marked defects in glycosphingolipid maturation within patient-derived brain organoids, indicating selective impairment of neural lipid glycosylation. Transcriptomic and biochemical analyses further revealed dysregulation of developmental pathways, including reduced Notch signalling and altered localization of both cleaved Notch1 and the Golgi-associated protein ACBD3. Consistent with impaired progenitor maintenance, COG5-deficient organoids displayed altered cleavage plane orientation in apical progenitors. Together, our findings suggest a link between Golgi organization, intracellular trafficking, membrane glycosylation, and lineage progression in human neural progenitors, identifying Golgi architecture as an important regulator of apical radial glial cell biology and cortical development.

cell biology↗

bioRxiv2026

LFQ Benchmark Dataset - Generation Beta: Assessing Modern Proteomics Instruments and Acquisition Workflows with High-Throughput LC Gradients

Recent advances in liquid chromatography-mass spectrometry (LC-MS) have accelerated the adoption of high-throughput workflows that deliver deep proteome coverage using minimal sample amounts. This trend is largely driven by clinical and single-cell proteomics, where sensitivity and reproducibility are essential. Here, we extend our previous benchmark dataset (PXD028735) using next-generation LC-MS platforms optimized for rapid proteome analysis. We generated an extensive DDA/DIA dataset using a human-yeast-E. coli hybrid proteome. The proteome sample was distributed across multiple laboratories together with standardized analytical protocols specifying two short LC gradients (5 and 15 min) and low sample input amounts. This dataset includes data acquired on four different platforms, and features new scanning quadrupole-based implementations, extending coverage across different instruments and acquisition strategies. Our comprehensive evaluation highlights how technological advances and reduced LC gradients may affect proteome depth, quantitative precision, and cross-instrument consistency. The release of this benchmark dataset via ProteomeXchange (PXD070049 and PXD071205), allows for the acceleration of cross-platform algorithm development, enhance data mining strategies, and supports standardization of short-gradient, high-throughput LC-MS-based proteomics.

bioinformatics↗

bioRxiv2025

Intrabody-guided synapse proteomics defines pyramidal neuron input architecture and uncovers early remodeling in a mouse model of Alzheimer's disease

Across their proximal and distal dendritic domains, pyramidal neurons (PNs) integrate inputs that differ in morphology and function. Hippocampal CA1 PNs are among the earliest affected neurons in Alzheimers disease (AD), but the molecular composition of their inputs and selective vulnerability remain poorly defined. We develop an intrabody-guided proximity-labeling strategy that targets the biotin ligase TurboID to endogenous postsynaptic scaffolds for cell-autonomous mapping of postsynaptic proteomes. Targeting PSD95 or Homer1 enables selective labeling of excitatory postsynaptic proteins in mouse CA1 PNs and resolves subsynaptic organization by comparing the two probes. Mapping the proteomes of major CA1 inputs uncovers a proximal-distal molecular logic that underlies their distinct properties. Applying this approach in the AppNL-G-F AD mouse model reveals an early signaling-driven phase of synaptic remodeling followed by a later translation-linked phase, with persistent downregulation of glutamatergic components. These results provide a molecular atlas of CA1 PN inputs and identify stage-specific mechanisms of synaptic vulnerability in early AD.

neuroscience↗

bioRxiv2025

LipidQMap - An Open-Source Tool for Quantitative Mass Spectrometry Imaging of Lipids

Mass spectrometry imaging (MSI) is a powerful tool in both basic and clinical research, enabling spatial visualization of biomolecules and drugs in tissue sections. However, factors influencing mass spectrometric response during imaging are increasingly recognized for their impact on apparent molecular distribution. Quantitative mass spectrometry imaging (qMSI) addresses this variability by incorporating analytical standards that undergo the same processes as analytes. While qMSI sample preparation protocols for omics-scale quantitative lipidomics are actively evolving, software solutions for downstream data processing remain scarce. Here, we introduce LipidQMap, the first open-source platform for processing omics-scale qMSI lipidomics data. LipidQMap applies one-point calibration, normalizing annotated lipid signals against class-specific standards on a pixel-by-pixel basis, and generates concentration heat maps in pmol/mm2. The software supports centroided data import, recalibration, and lipid identification using a built-in, user-modifiable lipid database. LipidQMap resolves Na/H adduct isobaric overlaps by leveraging sodiated-to-protonated adduct ratios of standards, validated across several MSI platforms using mouse brain sections. Type II isobaric overlaps are corrected using predicted isotopic patterns. Extensive validation demonstrates that LipidQMap is robust across MSI platforms and harmonizes qMSI data, enabling more accurate and reproducible spatial lipid quantification.

bioinformatics↗

bioRxiv2025

The molecular landscape of polycystic kidneys is marked by common alterations in purine metabolism

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Cysts develop through dedifferentiation of tubular epithelial cells, but the sequence of molecular events and their relative importance remain unclear. To address this gap in knowledge, 40 cysts from 4 ADPKD kidneys and 4 microcystic tissues were mapped on transcriptomic and histological level. Cyst were heterogenous and we identified 6 cystic subclusters with 2 deviations from the main trajectory, dependent on the rate of interstitial remodeling, inflammation and dedifferentiation. Loss of proximal tubular marker gene expression was more pronounced compared to those of other tubular segments. Altered expression of metabolic pathways was consistent among the cysts, which was further analyzed in human and mouse cell lines. Purine metabolism was similarly altered in all ADPKD cell lines, and its modulation with azathioprine suppressed cyst formation in vitro. In conclusion, by focusing on common altered pathways in cysts and cell models, we have identified purine metabolism as a novel potential target in ADPKD.

molecular biology↗

bioRxiv2024

Longitudinally cultured precision cut lung slices as a predictive ex vivo model for lung cancer

Exploring alternatives to in vivo models, this study validates Precision Cut Lung Slices (PCLS) as a viable ex vivo platform for lung cancer research. We established the prolonged viability and structural preservation of PCLS, essential for accurate drug response studies. Using Paclitaxel as a benchmark drug and a therapeutically promising silver nanoparticles in combination with immunotherapy, we conducted a pioneering comparative analysis of its therapeutic effects on PCLS against traditional in vivo models. Results revealed that PCLS closely mimics in vivo responses, demonstrating comparable drug efficacy in tumor growth inhibition. This direct comparison not only confirms the utility of PCLS in simulating real-world outcomes but also emphasizes its potential in reducing animal testing. By providing a reliable, ethical, and efficient alternative for lung cancer studies, PCLS could significantly enhance preclinical research and drug development, marking a critical step towards more humane and representative scientific investigations.

cancer biology↗

bioRxiv2023

Integrating multiple omics levels using the human protein complexome as a framework, a multi-omics study of inborn errors of metabolism

Proteins organize into functional assemblies that drive diverse biological activities. Leveraging a comprehensive dataset of manually curated annotations for the human protein complexome, we investigated biological perturbations at the protein complex level. Using proteomics and transcriptomics data from fibroblasts of patients with inborn errors of metabolism (IEM) and control samples, we globally mapped information onto complex subunits to discern affected processes. Across the patient cohort (consisting of organic acidaemias, fatty acid oxidation defects and mitochondrial respiratory chain defect IEMs), mitochondrial oxidative phosphorylation emerged as the most perturbed pathway, identified through proteomics datasets. Simultaneously, metabolomics highlighted significant regulation of phospholipids in patients with Fatty Acid and Mitochondrial IEM. Moreover, proteomics analysis also revealed the dysregulation of protein complexes involved in histone (de)acetylation, a finding validated through Western Blot analysis measuring histone acetylation levels. This introduces a novel epigenetic dimension to IEM and metabolic research, suggesting avenues for further exploration. Our study demonstrates a multiomics data integration concept that maps proteomics and transcriptomics data onto model organism complexomes. This integrative approach can be extended to metabolomics and lipidomics, associating information with complexes having metabolic functions, such as enzymatic complexes. This global strategy for identifying disease-relevant perturbations offers a systems-wide perspective on molecular-level physiological and pathological changes. Such insights are crucial for devising clinical intervention strategies and prioritizing druggable pathways and complexes. The presented methodology provides a foundation for future investigations, emphasizing the importance of integrating multiomics data to comprehensively understand cellular machinery alterations and facilitate targeted therapeutic approaches.

biochemistry↗

bioRxiv2023

Ancient genomic linkage couples metabolism with erythroid development

Generation of mature cells from progenitors requires tight coupling of differentiation and metabolism. During erythropoiesis, erythroblasts are required to massively upregulate globin synthesis then clear extraneous material and enucleate to produce erythrocytes1-3. Nprl3 has remained in synteny with the -globin genes for >500 million years4, and harbours the majority of the -globin enhancers5. Nprl3 is a highly conserved inhibitor of mTORC1, which controls cellular metabolism. However, whether Nprl3 itself serves an erythroid role is unknown. Here, we show that Nprl3 is a key regulator of erythroid metabolism. Using Nprl3-deficient fetal liver and adult competitive bone marrow - fetal liver chimeras, we show that NprI3 is required for sufficient erythropoiesis. Loss of Nprl3 elevates mTORC1 signalling, suppresses autophagy and disrupts erythroblast glycolysis and redox control. Human CD34+ progenitors lacking NPRL3 produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Finally, we show that the -globin enhancers upregulate NprI3 expression, and that this activity is necessary for optimal erythropoiesis. Therefore, the anciently conserved linkage of NprI3, -globin and their associated enhancers has enabled coupling of metabolic and developmental control in erythroid cells. This may enable erythropoiesis to adapt to fluctuating nutritional and environmental conditions.

cell biology↗

bioRxiv2023

HIF-1α-mediated mitochondrial-glycolytic reprogramming controls the transition of precursor to terminally exhausted T cells

Functional exhaustion of T cells in cancer and persistent infections is characterized by the upregulation of inhibitory receptors, the progressive decline in cytokine secretion and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex) with phenotypic features of memory T cells and a stem-like capacity to self-renew. However, the metabolic principles of Tpex maintenance and the regulatory circuits that control the exhaustion of their progeny remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics and metabolomic analyses, we here show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the T cell exhaustion program. At the molecular level, we found that diminished mitochondrial respiration and metabolic remodeling cause oxidative stress, which inhibits the proteasomal degradation of hypoxia inducible factor 1 alpha (HIF-1) in Tpex cells. HIF-1 mediates the transcriptional-glycolytic reprogramming of Tpex cells as an initial step towards terminal differentiation and functional exhaustion. Finally, we show that enhancing respiration by limiting the glycolytic activity of CAR T cells is a feasible metabolic intervention strategy to preserve the stemness of Tpex cells during chronic viral infection and cancer immunotherapy.

immunology↗

bioRxiv2023

13C tracer analysis reveals the landscape of metabolic checkpoints in human CD8+ T cell differentiation

Naive T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start proliferation and generation of effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells. Here we used 13C metabolomics and transcriptomics to study the metabolic dynamics of CD8+ T cells in their complete course of differentiation from naive over stem-like memory to effector cells. The quiescence of naive T cells was evident in a profound suppression of glucose oxidation and a decreased expression of ENO1, downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naive T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of T cell regulatory genes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which paradoxically was not linked to changes in mitochondrial abundance. We further investigated the metabolic phenotype of exhausted T cells, finding that decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of suppressive and quiescence-associated genes, including TXNIP and KLF2. Thus, these results identify multiple features critical for the metabolic reprogramming that supports quiescence, proliferation and effector function of CD8+ T cells during differentiation. Further, an impairment of the same processes in exhaustion suggests that targeting these control points may be useful for both modulation of differentiation and prevention of exhaustion.

immunology↗

bioRxiv2022

Effects of lactate, super-GDF9 and low oxygen tension during biphasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte-complex

In vitro maturation (IVM) is an alternative assisted reproductive technology (ART) with reduced hormone related side-effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a biphasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer; and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed a similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a biphasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.

developmental biology↗

bioRxiv2022

Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing Mcl-1-addicted cancer cells

Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). We previously demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a Bcl-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). These findings implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic Bcl-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. In this study, we observed that BAPTAi alone induced apoptosis in lymphoma cell models that were highly sensitive to S63845, an Mcl-1 antagonist. BAPTAi provoked a rapid decline in Mcl-1 protein levels by inhibiting mTORC1-driven MCL-1 translation. Overexpression of nondegradable Mcl-1 rescued BAPTAi-induced cell death. We further examined how BAPTAi diminished mTORC1 activity and found that BAPTAi impaired glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, an up to now unkown effect of BAPTAi. All aforementioned effects of BAPTAi were also elicited by a BAPTAi analog with low affinity for Ca2+. Thus, our work reveals PFKFB3 inhibition as an unappreciated Ca2+-independent mechanism by which BAPTAi impairs cellular metabolism and ultimately the survival of Mcl-1-dependent cancer cells. Our work has two important implications. First, direct inhibition of PFKFB3 emerged as a key regulator of mTORC1 activity and a promising target in the treatment of Mcl-1-dependent cancers. Second, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.

cell biology↗

bioRxiv2021

Species-specific mitochondria dynamics and metabolism regulate the timing of neuronal development

The evolution of species involves changes in the timeline of key developmental programs. Among these, neuronal development is considerably prolonged in the human cerebral cortex compared with other mammals, leading to brain neoteny. Here we explore whether mitochondria influence the species-specific properties of cortical neuron maturation. By comparing human and mouse cortical neuronal maturation at high temporal and cell resolution, we found a slower pattern of mitochondria development in human cortical neurons compared with the mouse, together with lower mitochondria metabolic activity, particularly oxidative phosphorylation. Stimulation of mitochondria metabolism in human neurons resulted in accelerated maturation, leading to excitable and complex cells weeks ahead of time. Our data identify mitochondria as important regulators of the pace of neuronal development underlying human-specific features of brain evolution.

neuroscience↗

bioRxiv2020

ANTIMETABOLIC COOPERATIVITY WITH THE CLINICALLY APPROVED KIDROLASE AND TYROSINE KINASE INHIBITORS TO ERADICATE CML STEM CELLS

Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective treatment for chronic myeloid leukemia (CML) and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells indicate that new therapeutic strategies are needed to prevent molecular relapses. We investigated the metabolic pathways responsible for CML surviving to Imatinib exposure and its potential therapeutic utility to improve the efficiency of TKI against CML stem cells. Using complementary cell-based techniques, we demonstrated that TKI suppressed glycolysis in a large panel of BCR-ABL1 + cell lines as well as in primary CD34+ stem-like cells from CML patients. However, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase without inducing predominant CML cell death. Clinically relevant concentrations of TKI render CML progenitors and stem cells susceptible to Kidrolase. The combination of TKI with L-asparaginase reactivated the intinsic apoptotic pathway leading to efficient CML cell death. Thus, targeting glutamine metabolism with the clinically-approved drug Kidrolase, in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating CML stem cells.

cancer biology↗

bioRxiv2020

Exostosin-1 Glycosyltransferase Regulates Endoplasmic Reticulum Architecture and Dynamics

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of GTP nucleotides. Among post-translational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on ER structure remains unclear. Here, we show that Exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multi-omics data and super-resolution imaging to characterize the broad effect of EXT1 inactivation, including ER shape-dynamics-function relationships in mammalian cells. We have observed that, inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated to ER network extension. Our findings suggest that EXT1 drives glycosylation reactions involving ER structural proteins and high-energy nucleotide sugars, which might also apply to other organelles.

cell biology↗

bioRxiv2018

AMP-activated protein kinase is essential for the maintenance of energy levels during synaptic activation

While accounting for 2% of the total body mass, the brain is the organ that consumes the most energy. Although it is widely acknowledged that neuronal energy metabolism is tightly regulated, the mechanism how neurons meet their energy demand to sustain synaptic transmission remains poorly studied. Here we provide substantial evidence that the AMP-activated protein kinase (AMPK) plays a leading role in this process. Our results show that following synaptic activation, AMPK activation is required to sustain neuronal energy levels particularly through mitochondrial respiration. Further, our studies revealed that this metabolic plasticity regulated by AMPK is required for the expression of immediate early genes, synaptic plasticity and memory formation. These findings are important in the context of neurodegenerative disorders, as AMPK deregulation as it is observed in Alzheimers disease, impairs the metabolic response to synaptic activation. Altogether, our data provides the proof of concept that AMPK is an essential player in the regulation of neuroenergetic metabolism plasticity induced in response to synaptic activation.

neuroscience↗