Biology subjects

Baekelandt, V.

Publications and source records attributed to Baekelandt, V..

12 recordsLinked to original sources

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

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

Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

BackgroundATP10B, a transmembrane lipid flippase located in late endosomes and lysosomes, facilitates the export of glucosylceramide and phosphatidylcholine by coupling this process to ATP hydrolysis. Recently, loss-of-function mutations in the ATP10B gene have been identified in Parkinsons disease patients, pointing to ATP10B as a candidate genetic risk factor. Previous studies have shown compromised lysosomal functionality upon ATP10B knockdown in human cell lines and primary cortical neurons. However, its role in vivo and specifically in the nigrostriatal dopaminergic system remains poorly understood. MethodsTo investigate the role ATP10B in PD neuropathology, we induced ATP10B knockdown specifically in substantia nigra pars compacta neurons of rats using viral vector technology. Two different microRNA-based shRNA constructs targeting distinct regions of the ATP10B mRNA were used to cross-validate the findings. Behavioral evaluation, dopamine transporter 18F-FE-PE2I positron emission tomography imaging and neuropathological examination of the nigrostriatal pathway at one year post-injection were conducted. Additionally, midbrain neuronal cultures derived from ATP10B knock-out human induced pluripotent stem cells clones were used to study the impact of ATP10B loss in dopaminergic neurons in a more translational model. ResultsATP10B knockdown in rat brain induced Parkinsonian motor deficits, and longitudinal striatal dopamine transporter 18F-FE-PE2I PET imaging revealed a progressive decrease in binding potential. Immunohistochemical analysis conducted one year post-injection confirmed the loss of dopaminergic terminals in the striatum, alongside a loss of dopaminergic neurons in the substantia nigra pars compacta. The expression of LAMP1, LAMP2a, cathepsin B and glucocerebrosidase was studied by immunofluorescence in the surviving dopaminergic neurons. A decrease in lysosomal numbers and an increase in lysosomal volume were observed more consistently in one of the knockdown constructs. The vulnerability of dopaminergic neurons to ATP10B loss-of-function was also observed in midbrain neuronal cultures derived from ATP10B knock-out human induced pluripotent stem cells clones, which showed a significant reduction in TH-positive neurons. ConclusionTaken together, our findings demonstrate that ATP10B depletion detrimentally impacts the viability of dopaminergic neurons both in vivo and in vitro. Moreover, a broader impact on the functionality of the nigrostriatal pathway was evidenced as rats with ATP10B knockdown exhibited motor impairments similar to those observed in PD patients.

neuroscience↗

bioRxiv2024

PAK6 promotes neuronal autophagy by regulating TFEB nuclear translocation.

Autophagy is a highly conserved homeostatic process essential for the bulk degradation of cytoplasmic components and aggregated proteins. Multiple evidence indicates that impairment of (macro)autophagy leads to neurodegeneration, such as Parkinson disease (PD). Our previous work showed that p21 activated kinase 6 (PAK6) interacts with the PD-associated leucine-rich repeat kinase (LRRK2) to promote neurite outgrowth in the mouse striatum; still the function of PAK6 in the brain is largely unknown. Here, we found that downregulation of neuronal but not glial mbt, the D. melanogaster homolog of PAK6, impairs autophagy-lysosomal function. PAK6 overexpression in cells and in C. elegans increases transcription factor EB (TFEB) nuclear translocation in a kinase activity-dependent manner. Mechanistically, PAK6 forms a complex with TFEB to regulate its nuclear localization in a manner dependent on phosphorylation of and binding to 14-3-3 proteins and phosphorylation of TFEB at S467. In line with its ability to promote neuronal autophagy, mbt downregulation exacerbates alpha-synuclein toxicity in Drosophila dopaminergic neurons. Moreover, PAK6 overexpression in the substantia nigra of mutant LRRK2 mice reduces the burden of phosphorylated alpha-synuclein in dopaminergic neurons. Altogether, our study uncovers a novel role of PAK6 as a positive regulator of autophagy via TFEB and suggests that modulating its activity may represent a way to selectively turn on autophagy in neurons, with implications for the treatment of neurodegenerative disorders.

neuroscience↗

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↗

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↗

bioRxiv2020

Rit2 reduces LRRK2 kinase activity and protects against alpha-synuclein neuropathology

In Parkinsons disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear but hypothesized to involve the autophagy-lysosome pathway (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, hyperactivate kinase activity and its pharmacological inhibition reduces pS129-aSyn inclusions. We observed selective downregulation of the novel PD risk factor RIT2 in G2019S-LRRK2 expressing cells. Here we studied whether RIT2 could modulate LRRK2 kinase activity. RIT2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of RIT2 conferred neuroprotection against AAV-A53T-aSyn. Furthermore, RIT2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. Our data indicate that RIT2 inhibits overactive LRRK2 to ameliorate ALP impairment and counteract aSyn aggregation and related deficits. Targeting RIT2 could represent a novel strategy to combat neuropathology in familial and idiopathic PD.

neuroscience↗

bioRxiv2020

a-Synuclein strains influence multiple system atrophy via central and peripheral mechanisms

Multiple system atrophy (MSA) is a progressive neurodegenerative disease with prominent autonomic and motor features. Different disease subtypes are distinguished by their predominant parkinsonian or cerebellar signs. The pathognomonic feature of MSA is the presence of -synuclein (Syn) protein deposits in glial cells of the central and peripheral nervous system. It is unclear why MSA, that invariably presents with Syn pathology, is clinically so heterogeneous, why it progresses at varying rates and how neuroinflammation affects disease progression. Recently, it was shown that different strains of Syn can assemble in unique disease environments but also that a variety of strains might exist in the brain of MSA patients. We therefore investigated if different Syn strains might influence MSA disease progression. To this aim, we injected two recombinant strains of Syn in MSA transgenic mice and found that they significantly impact MSA disease progression in a strain-dependent way via oligodendroglial, neurotoxic and immune-related mechanisms. Neurodegeneration and brain atrophy were accompanied by unique microglial and astroglial responses and the recruitment of central and peripheral immune cells. The differential activation of microglial cells correlated with the structural features of Syn strains both in vitro and in vivo. By injecting Syn strains in MSA mice we could more closely mimic a comprehensive MSA phenotype in an experimental setting. This study therefore shows that i) MSA phenotype is governed by both the Syn strain nature and the host environment and ii) Syn strains can directly trigger a detrimental immune response related to disease progression in MSA.

neuroscience↗

bioRxiv2019

Kinase inhibition of G2019S-LRRK2 restores autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions

The Parkinsons disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a potent modulator of autophagy and impacts on lysosome biology and function, but unclarity exists on the precise mechanics of its role and the direction of this modulation. LRRK2 is also involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and most LRRK2 familial cases manifesting with Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to reconcile these independent pieces of information to obtain a comprehensive knowledge on LRRK2-associated pathology that could also be generalized to the idiopathic disease.\n\nHere, we report a focused investigation on the role of PD-causing G2019S-LRRK2 in the autophagy-lysosome pathway in a recombinant cell line model. Initially, we evaluated the effect of LRRK2 expression on autophagy-related transcriptome. Then, we found that G2019S-LRRK2 leads to accumulation of autophagosomes with no net effect on autophagy induction. This is linked to abnormalities in lysosome morphology and proteolytic activity that are associated with a decrease in the successful formation of autolysosomes. Despite some of these features being shared by WT-LRRK2, alpha-synuclein intracellular inclusions are specifically found in G2019S-LRRK2 cells. Pharmacological kinase inhibition is capable of rescuing defects in the autophagy-lysosome pathway and reducing the number of inclusions. Notably, this effect is prevented by upstream blockade of autophagosome-lysosome fusion events, highlighting this step of the process as critical for alpha-synuclein clearance.

cell biology↗