Biology subjects

Van den Haute, C.

Publications and source records attributed to Van den Haute, C..

11 recordsLinked to original sources

bioRxiv2026

A druggable ATP13A3-antizyme switch controls adaptive polyamine uptake in cancer

Cellular polyamine depletion is a promising anticancer strategy, but compensatory polyamine uptake limits efficacy when synthesis is blocked by DFMO (difluoromethylornithine), a clinically approved inhibitor of ornithine decarboxylase. The transporter and feedback mechanism driving this adaptive response have remained unclear. Despite their similar biochemical properties, we identify ATP13A3, rather than ATP13A2, as the principal DFMO-responsive polyamine importer, suggesting that these isoforms regulate distinct polyamine fluxes. Mechanistically, the polyamine sensor antizyme not only restrains polyamine biosynthesis but also selectively inhibits ATP13A3-mediated uptake, a brake that is relieved upon DFMO treatment. This regulatory circuit exposes distinct polyamine-acquisition states across cancers, defining synthesis- and/or uptake-biased subtypes that can shift during disease progression. Melanoma metastasis and vemurafenib resistance evolve toward increased ATP13A3-dependent uptake. The polyamine uptake branch controlled by ATP13A3-antizyme regulation can be pharmacologically blocked by AMXT 1501, which directly inhibits ATP13A3. Together, our findings explain DFMO adaptation through ATP13A3-antizyme control and establish ATP13A3 as a targetable node for polyamine depletion strategies in multiple cancers, supporting ongoing clinical evaluation of combined DFMO/AMXT 1501 therapy.

cell biology↗

bioRxiv2026

Protein aggregation inhibitors induce divergent transcriptional responses in a cellular model of a-synuclein seeded aggregation

Parkinsons disease (PD), dementia with Lewy Bodies (DLB) and multiple system atrophy (MSA) are progressive neurodegenerative disorders marked by the pathological aggregation of alpha-synuclein ([a]Syn). Despite significant research efforts, effective therapeutic interventions remain elusive due to limited understanding of the cellular effects of [a]Syn aggregation and propagation. This study presents the development of a scalable cellular seeding assay for screening small molecules targeting cellular [a]Syn seeded aggregation. By leveraging a fluorescent reporter of [a]Syn and phenotypic screening, the assay enables high-throughput evaluation of potential inhibitors in a cellular environment mimicking disease pathology. We evaluated three different Syn aggregation inhibitors tested in clinical trials for PD: Minzasolmin, Emrusolmin and EGCG and profiled gene expression using multiplexed single cell RNA sequencing in order to examine their distinct effects on cellular pathways associated with [a]Syn overexpression or seeded aggregation. Two cellular activities were prominently affected: lipid metabolism and rRNA processing. Notably, while EGCG effects were confined to cells with aggregated Syn, Minzasolmin and Emrusolmin also produced transcriptional changes in cells without aggregated Syn. Each of the compounds tested induced a partial reversal of transcriptional effects resulting from Syn seeded aggregation. We identified 391 genes that were no longer significantly differentially expressed upon addition of compound, relative to cells with seeded aggregation. This platform bridges phenotypic screening and molecular pathway analysis, providing insights into druggable pathways for synucleinopathies. The molecular signatures identified here can assist in testing and benchmarking future drug discovery leads.

neuroscience↗

bioRxiv2025

From injury to recovery: Functional neuronal regeneration after traumatic brain injury in the telencephalon of the young adult African turquoise killifish

Neuronal loss caused by neurodegenerative diseases and traumatic brain injuries (TBI) often results in long-term disabilities, highlighting the urgent need for further research and effective regenerative strategies. In mammals, neurogenic capacity is inherently limited and declines further with age. In contrast, the young adult killifish demonstrates a remarkable ability to regenerate neurons in the telencephalon following TBI. However, it remains unknown whether and when these newly generated neurons functionally integrate into existing circuits, as traditional histological analysis of fixed tissue offers only static insights into this dynamic process. To this end, we optimized a retroviral vector strategy to label dividing stem cells and their progeny, including newborn neurons. By introducing a combination of novel approaches i.e., retroviral vector labeling, electrophysiology and a conditioned place avoidance test, we investigated the generation, morphology, and synaptic integration of newborn neurons following TBI in the dorsomedial (Dm) zone of the telencephalon, a region homologous to the mammalian amygdala in other teleost fish. Our results show that injury-induced adult-born neurons functionally integrate into existing circuits, and that killifish can achieve functional behavioral recovery after TBI. While previous histological assessments using a stab-wound injury suggested a 30-day recovery period, our functional data reveal that full behavioral recovery requires approximately 50 days. At this point, fish successfully relearn to avoid a conditioned place, and the new neurons exhibit mature morpho-electric characteristics, including abundant dendritic spines. Electrophysiological analysis revealed that newborn neurons in an injured environment take longer to mature when compared to neurons in naive killifish. Together, our findings demonstrate that structural regeneration aligns with functional recovery, and establish retroviral vectors as a powerful tool for birth dating injury-induced neurogenesis in teleosts. Killifish thus represent a promising model for studying interventions aimed at enhancing neuronal maturation and integration after brain injury. Key pointsO_LIA retroviral vector strategy allows specific and sparse labeling of adult-born neurons in the killifish brain. C_LIO_LIThe Dm zone in the telencephalon of the killifish is responsible for avoidance learning and memory and thus homologous to the mammalian amygdala. C_LIO_LIIn young adult killifish, upon Dm injury, adult-born neurons mature morphologically and functionally in 50 days, which is slower than in constitutive neurogenesis. C_LIO_LIThe timing and extent of behavioral recovery from such injury aligns with morpho-electric observations. C_LI

neuroscience↗

bioRxiv2025

Development of convolutional neural networks for automated brain-wide histopathological analysis in mouse models of synucleinopathies

Preclinical animal models are indispensable for uncovering disease mechanisms and developing novel therapeutic interventions in synucleinopathies. Key readouts including neuronal cell death, neuroinflammation and alpha-synuclein protein aggregation, are routinely assessed by histological methods. However, traditional characterization of histological samples is labor-intensive and time-consuming. There is a growing need for reproducible and high-throughput tools to capture region- and cell type-specific changes, ultimately improving the predictive value of preclinical studies. To address this, our study introduces a pipeline using convolutional neural networks (CNNs) for high-throughput, unbiased analysis of immunohistological data in mouse brains. We have trained five CNN-based models to autonomously identify brain regions and detect markers of neurodegeneration, neuroinflammation, and alpha-synuclein aggregation. These models provide accurate, region-specific insights at cellular resolution without manual annotation, significantly speeding up analysis time from weeks to minutes. Our approach enhances the precision and efficiency of histological assessments, providing robust, brain-wide results in various animal models of synucleinopathies.

neuroscience↗

bioRxiv2024

The polyamine transporter ATP13A3 mediates DFMO-induced polyamine uptake in neuroblastoma

High-risk neuroblastomas, often associated with MYCN oncogene amplification, are addicted to polyamines, small polycations vital for cellular functioning. We have shown that neuroblastoma cells increase polyamine uptake when exposed to the polyamine biosynthesis inhibitor DFMO, currently in clinical trial, and that this mechanism limits the efficacy of the drug. While this finding resulted in the clinical development of polyamine transport inhibitors including AMXT 1501, presently under clinical investigation in combination with DFMO, the mechanisms and transporters involved in DFMO-induced polyamine uptake are unknown. Knockdown of ATP13A3, a member of the P5B-ATPase family, limited basal and DFMO-induced polyamine uptake, attenuated MYCN-amplified and non-MYCN-amplified neuroblastoma cell growth and potentiated the inhibitory effects of DFMO. Overexpression of ATP13A3 in neuroblastoma cells increased polyamine uptake, which was inhibited by AMXT 1501, highlighting ATP13A3 as a key target of the drug. The association between high ATP13A3 expression and poorer survival in neuroblastoma further supports a role of this transporter in neuroblastoma progression. Thus, this study identified ATP13A3 as a critical regulator of basal and DFMO-induced polyamine uptake and a novel therapeutic target for neuroblastoma. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/581161v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@11bef78org.highwire.dtl.DTLVardef@106c250org.highwire.dtl.DTLVardef@bf39b1org.highwire.dtl.DTLVardef@64d05f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

ATP13A3 Variants Promote Pulmonary Arterial Hypertension by Disrupting Polyamine Transport

AimsPotential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in pulmonary arterial hypertension (PAH) patients. ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. Here, we sought to determine the biological function of ATP13A3 in vascular endothelial cells and how PAH-associated mutations may contribute to disease pathogenesis. We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH. Methods and ResultsWe studied the impact of ATP13A3 deficiency and overexpression in endothelial cell (EC) models (human pulmonary ECs, blood outgrowth ECs (BOECs) and HMEC-1 cells), including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). ATP13A3 localised to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content, consistently reduced putrescine uptake, and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake, with an overall preference of putrescine > spermidine > spermine. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation and increased monolayer permeability to thrombin. Assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germ-line Atp13a3 frameshift variant representing a human mutation spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling and muscularisation of pulmonary vessels. ConclusionWe identify ATP13A3 as a polyamine transporter, deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH. Translational perspectiveRare missense ATP13A3 disease-associated variants have been identified in patients with pulmonary arterial hypertension (PAH), though their pathogenicity has not been confirmed as the function of ATP13A3 is not known. We have identified ATP13A3 as a polyamine transporter, showing that ATP13A3 deficiency impaired polyamine homeostasis and uptake, and drove endothelial dysfunction. Conversely, overexpression increased polyamine uptake and rescued the proapoptotic phenotype of cells harbouring a disease-associate variant. Mice heterozygous for a disease-associated Atp13a3 mutation spontaneously develop PAH. These findings support the rationale for exploring dysregulated polyamine homeostasis in PAH and suggest a potential for therapeutic targeting of polyamine pathways in PAH.

cell biology↗

bioRxiv2023

Functional characterization of ATP13A2 variants associated with distinct neurodegenerative disorders

ATP13A2 is a late endolysosomal transporter that exports the polyamines spermine and spermidine from the organellar lumen to the cytosol. Loss-of-function variants in ATP13A2 are causative for Kufor-Rakeb syndrome (KRS, a recessive juvenile-onset parkinsonism with dementia) and have also been identified in early-onset PD (EOPD) and hereditary spastic paraplegia (HSP). Furthermore, candidate pathogenic ATP13A2 variants have been identified in neuronal ceroid lipofuscinosis (NCL; M854R), multiple system atrophy (MSA; Y1020C) and amyotrophic lateral sclerosis (ALS; I411M) suggesting that ATP13A2 may be implicated in a broader range of neurodegenerative disorders. Since the functional consequences of the NCL, MSA, and ALS variants have not yet been examined, we here characterized these ATP13A2 variants in terms of subcellular localization, cellular polyamine uptake, and transport activity. We found that the homozygous NCL-associated M854R variant results in an instable protein with low expression levels, leading to complete loss of ATPase and cellular polyamine uptake activity. The heterozygous MSA-linked Y1020C variant is properly localized and presents only partially decreased ATPase activity without affecting cellular polyamine uptake. The ALS-associated I411M variant is also correctly localized and exhibits a minor effect on cellular polyamine uptake, however, without a significant impact on ATPase activity. Taken together, only the homozygous NCL variant of ATP13A2 causes a complete loss-of-function, validating that ATP13A2 dysfunction is implicated in NCL. The ALS and MSA variants only presented a subtle functional defect, questioning whether these heterozygous variants are pathogenic and whether ATP13A2 dysfunction may cause MSA or ALS.

molecular biology↗

bioRxiv2023

The lipid flippase ATP10B enables cellular lipid uptake under stress conditions

Pathogenic ATP10B variants have been described in patients with Parkinsons disease and dementia with Lewy body disease, and we previously established ATP10B as a late endo-/lysosomal lipid flippase transporting both phosphatidylcholine (PC) and glucosylceramide (GluCer) from the lysosomal exoplasmic to cytoplasmic membrane leaflet. Since several other lipid flippases regulate cellular lipid uptake, we here examined whether also ATP10B impacts cellular lipid uptake. Transient co-expression of ATP10B with its obligatory subunit CDC50A stimulated the uptake of fluorescently (NBD-) labeled PC in HeLa cells. This uptake is dependent on the transport function of ATP10B, is impaired by disease-associated variants and appears specific for NBD-PC. Uptake of non-ATP10B substrates, such as NBD-sphingomyelin or NBD-phosphatidylethanolamine is not increased. Remarkably, in stable cell lines co-expressing ATP10B/CDC50A we only observed increased NBD-PC uptake following treatment with rotenone, a mitochondrial complex I inhibitor that induces transport-dependent ATP10B phenotypes. Conversely, Im95m and WM-115 cells with endogenous ATP10B expression, present a decreased NBD-PC uptake following ATP10B knockdown, an effect that is exacerbated under rotenone stress. Our data show that the endo-/lysosomal lipid flippase ATP10B contributes to cellular PC uptake under specific cell stress conditions.

cell biology↗

bioRxiv2023

ATP13A4 upregulation drives the elevated polyamine transport system in the breast cancer cell line MCF7

Polyamine homeostasis is disturbed in several human diseases, including cancer, which is hallmarked by increased intracellular polyamine levels and an upregulated polyamine transport system (PTS). So far, the polyamine transporters contributing to the elevated levels of polyamines in cancer cells have not yet been described, despite the fact that polyamine transport inhibitors are considered for cancer therapy. Here, we tested whether upregulation of candidate polyamine transporters of the P5B- transport ATPase family is responsible for the increased PTS in the well-studied breast cancer cell line MCF7 compared to the non-tumorigenic epithelial breast cell line MCF10A. We found that MCF7 cells present elevated expression of a previously uncharacterized P5B-ATPase ATP13A4, which is responsible for the elevated polyamine uptake activity. Furthermore, MCF7 cells are more sensitive to polyamine cytotoxicity, as demonstrated by cell viability, cell death and clonogenic assays. Importantly, overexpression of ATP13A4 WT in MCF10A cells induces a MCF7 polyamine phenotype, with significantly higher uptake of BODIPY-labelled polyamines and increased sensitivity to polyamine toxicity. In conclusion, we establish ATP13A4 as a new polyamine transporter in the human PTS and show that ATP13A4 may play a major role in the increased polyamine uptake of breast cancer cells. ATP13A4 therefore emerges as a candidate therapeutic target for anticancer drugs that block the PTS.

cell biology↗

bioRxiv2022

Novel green fluorescent polyamines to analyze ATP13A2 and ATP13A3 activity in the mammalian polyamine transport system

Cells acquire the polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary action of polyamine uptake and synthesis pathways. The endosomal P5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2 and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than ATP13A2. We also compared the uptake of new green-fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P5B-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure-activity relationship (SAR) suggesting that isoform-specific polyamine probes can be designed.

biochemistry↗

bioRxiv2021

Label-free imaging of membrane potentials by intramembrane field modulation, assessed by Second Harmonic Generation Microscopy

Optical interrogation of cellular electrical activity has proven itself essential for understanding cellular function and communication in complex networks. Voltage-sensitive dyes are important tools for assessing excitability but these highly lipophilic sensors may affect cellular function. Label-free techniques offer a major advantage as they eliminate the need for these external probes. In this work, we show that endogenous second harmonic generation (SHG) from live cells is highly sensitive to changes in membrane potential. Simultaneous electrophysiological control of a living (HEK293T) cell, through whole-cell voltage clamp reveals a linear relation between the SHG intensity and membrane voltage. Our results suggest that due to the high ionic strengths and fast optical response of biofluids, membrane hydration is not the main contributor to the observed field sensitivity. We further provide a conceptual framework that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero [Formula] tensor. Changing the membrane potential without surface modifications such as electrolyte screening offers high optical sensitivity to membrane voltage (~40% per 100 mV), indicating the power of SHG for label-free read-out. These results hold promise for the design of a non-invasive label-free read-out tool for electrogenic cells.

biophysics↗