Biology subjects

Lopes, F. M.

Publications and source records attributed to Lopes, F. M..

8 recordsLinked to original sources

bioRxiv2025

Human pluripotent stem cell-derived macrophages modify development of human kidney organoids

IntroductionThe human fetal kidney contains macrophages, innate immune cells postulated to enhance its development. Macrophages have also been implicated in the pathobiology of human kidney malformations and Wilms tumour. Human pluripotent stem cell (hPSC)-derived kidney organoids contain nephrons differentiated from intermediate mesoderm-like precursors. Endothelia are present between organoid tubules but fail to efficiently populate glomeruli. These organoids lack macrophages, as expected because in vivo kidney macrophages invade from yolk sac and liver. MethodsWe hypothesised that combining hPSC-derived macrophages with hPSC-derived kidney precursors modifies nephrogenesis. Macrophages harvested at early or later maturation stages were added to kidney precursors in numbers of 1%, 5% or 20% compared with constant numbers of nephrogenic cells. ResultsNo macrophages, as assessed by CD68 immunostaining, were detected in organoids without added macrophages. In contrast, composite organoids contained macrophages located between tubules, mimicking native human fetal kidneys. Quantification of tissue macrophages at the end of 18-day organoid culture positively correlated with the numbers of macrophages added. Assessed by CD31/PECAM-1 and CD68 co-immunostaining, some macrophages were near vessels but added macrophages neither increased the proportion of vessels nor endothelial invasion of glomeruli. Early-stage macrophages significantly increased the percentage area occupied by glomeruli, as assessed by synaptopodin immunostaining. The highest macrophage numbers inhibited overall growth, assessed by organoid area, additionally generating dysmorphic tissue when later stage macrophages were added. ConclusionDepending on their maturation stage and quantity, macrophages have beneficial or harmful effects on organoids. This supports the proposition that macrophages play roles in normal and abnormal development of human kidneys.

developmental biology↗

bioRxiv2025

A CRISPR-CAS9 high throughput machine-learning platform for modulation of genes involved in Parkinson's disease-associated PINK1-mitophagy in iPSC-derived dopaminergic neurons

Parkinsons disease (PD) is a progressive neurodegenerative disorder characterised by the loss of dopaminergic neurons, driven by complex molecular mechanisms that are not fully understood. To address this issue, we have developed a novel high-content phenotypic screening platform using human induced pluripotent stem cell-derived dopaminergic neurons to investigate the PINK1-PARKIN mitophagy pathway, a critical process in PD pathogenesis. Utilising high throughput, 384 well arrayed CRISPR-CAS9 genetic manipulation and high-content immunofluorescence imaging complemented with machine learning analysis, we examined ubiquitin (Ub) pSer65 levels. Ub pSer65, a potential PD clinical biomarker, is a key marker of mitophagy initiation in dopaminergic neurons upon mitophagy initiation using exogenous stimuli to mimic the disease relevant environment. The CRISPR-CAS9 knockout (KO) screen revealed two distinct phenotypic classes: essential genes causing cell death upon deletion, and genes modulating Ub pSer65 levels. Notably, KO of PINK1, PARKIN, and TOM7 genes decreased Ub pSer65 upregulation during mitophagy activation, confirming their established roles in the pathway and validating the suitability of the platform for target identification. This innovative platform provides a precise tool to further interrogate PD-associated genes, offering insights into mitophagy-related pathogenic mechanisms and identification of potential therapeutic targets. By bridging functional genomics with disease-specific neuronal models, this approach presents a promising strategy for advancing PD research and developing targeted interventions. To our knowledge, this is the first reported use of a human, translationally relevant cell model to study genetic perturbation within a disease relevant phenotype.

neuroscience↗

bioRxiv2023

Human HPSE2 gene transfer ameliorates bladder pathophysiology in a mutant mouse model of urofacial syndrome.

Rare early onset lower urinary tract disorders include defects of functional maturation of the bladder. Current treatments do not target the primary pathobiology of these diseases. Some have a monogenic basis, such as urofacial, or Ochoa, syndrome (UFS). Here, the bladder does not empty fully because of incomplete relaxation of its outflow tract, and subsequent urosepsis can cause kidney failure. UFS is associated with biallelic variants of HPSE2, encoding heparanase-2. This protein is detected in pelvic ganglia, autonomic relay stations that innervate the bladder and control voiding. Bladder outflow tracts of Hpse2 mutant mice display impaired neurogenic relaxation. We hypothesized that HPSE2 gene transfer soon after birth would ameliorate this defect and explored an adeno-associated viral (AAV) vector-based approach. AAV9/HPSE2, carrying human HPSE2 driven by CAG, was administered intravenously into neonatal mice. In the third postnatal week, transgene transduction and expression were sought, and ex vivo myography was undertaken to measure bladder function. In mice administered AAV9/HPSE2, the viral genome was detected in pelvic ganglia. Human HPSE2 was expressed and heparanase-2 became detectable in pelvic ganglia of treated mutant mice. On autopsy, wild-type mice had empty bladders whereas bladders were uniformly distended in mutant mice, a defect ameliorated by AAV9/HPSE2 treatment. Therapeutically, AAV9/HPSE2 significantly ameliorated impaired neurogenic relaxation of Hpse2 mutant bladder outflow tracts. Impaired neurogenic contractility of mutant detrusor smooth muscle was also significantly improved. These results constitute first steps towards curing UFS, a clinically devastating genetic disease featuring a bladder autonomic neuropathy. SummaryIn the first gene therapy for genetic bladder disease, we cured autonomic neurons using AAV-mediated gene delivery in a mouse model of urofacial syndrome.

neuroscience↗

bioRxiv2023

Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human ESCs and iPSCs reprogrammed from a family with HNF1B-asscociated DKMs. Mutant organoids contained enlarged malformed tubules and displayed deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cAMP-mediated dilatation seen in controls. Bioinformatic analyses indicated abnormal WNT, calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 was upregulated in mutant organoids and was detected in their tubules and in fetal human DKM dysplastic epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule morphogenesis and functional differentiation. They additionally suggest druggable targets to ameliorate disease.

developmental biology↗

bioRxiv2023

Lipofuscin-like autofluorescence within microglia and its impact on studying microglial engulfment

Engulfment of cellular material and proteins is a key function for microglia, a resident macrophage of the central nervous system (CNS). Among the techniques used to measure microglial engulfment, confocal light microscopy has been used the most extensively. Here, we show that autofluorescence (AF), likely due to lipofuscin and typically associated with aging, can also be detected within microglial lysosomes in the young mouse brain by light microscopy. This lipofuscin-AF signal accumulates first within microglia and increases with age, but it is not exacerbated by amyloid beta-related neurodegeneration. We further show that this lipofuscin-AF signal within microglia can confound the interpretation of antibody-labeled synaptic material within microglia in young adult mice. Finally, we implement a robust strategy to quench AF in mouse, marmoset, and human brain tissue.

neuroscience↗

bioRxiv2020

Evidence-based gene expression modulation correlates with transposable element knock-down

BackgroundTransposable elements (TEs) are major components of plant genomes. Despite being regarded as "junk DNA" at first, TEs play important roles for the organisms they are found in. The most obvious and easily recognizable effects caused by TEs result from their mobility, which can disrupt coding sequences or promoter regions. However, with the recent advances in transcriptomics, it is becoming increasingly evident that TEs can act as an additional layer of gene expression regulation through a number of processes, which can involve production of non-coding RNAs. Here, we describe how Tnt1, a stress-responsive LTR-retrotransposon, interferes with gene expression and modulate a number of developmental aspects in tobacco. ResultsThrough an RNAi approach, we generated tobacco (HP) lines knocked-down for Tnt1 expression. Quantitative RT-PCR experiments confirm that Tnt1 is downregulated in HP lines after ethylene exposure. A RNA-seq experiment was performed and through two independent bioinformatic approaches (with different stringencies) we found 932 and 97 differentially expressed genes in HP lines. A number of phenotypes were observed in such lines, namely lesion mimicry in leaves, underdevelopment of the root system, overproduction of root hairs and early loss of seed viability. Folding prediction of part of the Tnt1 mRNA reveals putative stem-loop secondary structures containing transcriptional regulation sequences, suggesting it could be a source of small RNAs. We also propose a model to explain the Tnt1 expression in both homeostatic and stress conditions, and how it could interact with stress-responsive genes. ConclusionsOur results are consistent that interferences with Tnt1 transcript levels correlate with transcriptomic and phenotypic changes, suggesting a functional role for this element during plant development and stress response.

molecular biology↗

bioRxiv2020

Feature Extraction Approaches for Biological Sequences: A Comparative Study of Mathematical Models

The number of available biological sequences has increased significantly in recent years due to various genomic sequencing projects, creating a huge volume of data. Consequently, new computational methods are needed to analyze and extract information from these sequences. Machine learning methods have shown broad applicability in computational biology and bioinformatics. The utilization of machine learning methods has helped to extract relevant information from various biological datasets. However, there are still several obstacles that motivate new algorithms and pipeline proposals, mainly involving feature extraction problems, in which extracting significant discriminatory information from a biological set is challenging. Considering this, our work proposes to study and analyze a feature extraction pipeline based on mathematical models (Numerical Mapping, Fourier, Entropy, and Complex Networks). As a case study, we analyze Long Non-Coding RNA sequences. Moreover, we divided this work into two studies, e.g., (I) we assessed our proposal with the most addressed problem in our review, e.g., lncRNA vs. mRNA; (II) we tested its generalization on different classification problems, e.g., circRNA vs. lncRNA. The experimental results demonstrated three main contributions: (1) An in-depth study of several mathematical models; (2) a new feature extraction pipeline and (3) its generalization and robustness for distinct biological sequence classification.

bioinformatics↗

bioRxiv2020

Oxidative switch drives mitophagy defects in dopaminergic parkin mutant patient neurons

Background Mutations in parkin are the most common cause of early onset Parkinsons disease. Parkin is an E3 ubiquitin ligase, functioning in mitophagy. Mitochondrial abnormalities are present in parkin mutant models. Patient derived neurons are a promising model in which to study pathogenic mechanisms and therapeutic targets. Here we generate induced neuronal progenitor cells from parkin mutant patient fibroblasts with a high dopaminergic neuron yield. We reveal changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Methods Fibroblasts from 4 controls and 4 parkin mutant patients were transformed into induced neuronal progenitor cells and subsequently differentiated into dopaminergic neurons. Mitochondrial morphology, function and mitophagy were evaluated using live cell fluorescent imaging, cellular ATP and reactive oxygen species production quantification. Results Direct conversion of control and parkin mutant patient fibroblasts results in induced neuronal progenitor and their differentiation yields high percentage of dopaminergic neurons. We were able to observe changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Our results show that when pre-neurons are glycolytic early in differentiation mitophagy is unimpaired by PRKN deficiency. However as neurons become oxidative phosphorylation dependent, mitophagy is severely impaired in the PRKN mutant patient neurons. These changes correlate with changes in mitochondrial function and morphology; resulting in lower neuron yield and altered neuronal morphology. Conclusions Induced neuronal progenitor cell conversion can produce a high yield of dopaminergic neurons. The mitochondrial phenotype, including mitophagy status, is highly dependent on the metabolic status of the cell. Only when neurons are oxidative phosphorylation reliant the extent of mitochondrial abnormalities are identified. These data provide insight into cell specific effects of PRKN mutations, in particular in relation to mitophagy dependent disease phenotypes and provide avenues for alternative therapeutic approaches.

neuroscience↗