Biology subjects

Bessa, J.

Publications and source records attributed to Bessa, J..

12 recordsLinked to original sources

bioRxiv2026

A motor endurance assay for zebrafish larvae: proof of principle in chemical and genetic models of neurological disease

Motor fatigability is an early feature of neurological disease, including amyotrophic lateral sclerosis (ALS), but motor endurance assays for zebrafish are largely restricted to juvenile or adult animals. Here, we developed a gravity-fed counter-current swimming assay to quantify motor endurance in 5-7 days post-fertilisation (dpf) zebrafish larvae, which supports the parallel testing of multiple larvae. We detected an increase in motor endurance induced by exposure to caffeine, and a reduction induced by the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP+). In these tests, motor endurance showed a greater dynamic range than spontaneous locomotion. Further, we generated and tested transgenic zebrafish larvae with neuron-restricted overexpression of human TDP43 (hTDP43), the major pathological protein in ALS patients. Zebrafish with neuronal hTDP43 expression showed ALS-relevant hTDP43 mislocalisation and significantly reduced motor endurance at 5 dpf. This method for motor endurance assessment in 5-7 dpf zebrafish is simple, sensitive, and enables the characterisation of motor fatigability in models of human neurological disease (TDP43-linked ALS and MPP+-induced parkinsonism).

neuroscience↗

bioRxiv2026

A dual-phase enhancer couples progenitor maintenance and pancreatic lineage stability

Enhancers orchestrate transcriptional programs that control organ development and maintain differentiated cell states, yet how individual enhancers integrate developmental and long-term tissue maintenance logic remains poorly understood. Here, we identify a distal enhancer downstream of ptf1a (z3-DpE) as a regulatory node coupling pancreatic development with acinar cell homeostasis in zebrafish. Deletion of z3-DpE reduces ptf1a expression in pancreatic multipotent progenitor cells (MPCs), leading to depletion of the progenitor pool, altered morphogenesis, and premature exocrine differentiation. Transcriptomic analysis reveals broad repression of proliferation- and morphogenesis-related genes, including Notch pathway components essential for progenitor maintenance. After differentiation, loss of z3-DpE contributes to acinar cell loss, expansion of ductal and endocrine compartments, and disrupted pancreatic architecture. Chromatin-accessibility profiling of purified acinar cells reveals that reduced ptf1a activity leads to widespread remodeling of the acinar chromatin landscape, with decreased accessibility at loci associated with acinar identity and developmental programs, and increased accessibility at sites linked to inflammation, epithelial plasticity, and pancreatic cancer susceptibility. Histopathological analysis shows disorganized acinar tissue with increased duct-like structures and mucinous lesions reminiscent of early pancreatic neoplasia. Thus, z3-DpE safeguards acinar identity by sustaining ptf1a expression and a chromatin landscape that restricts fate instability and pathological plasticity. Our findings demonstrate the mechanistic sufficiency of a single enhancer to coordinate progenitor expansion and long-term lineage stabilization, providing a paradigm for how a developmental regulatory element is redeployed to preserve tissue integrity and suppress disease-associated plasticity.

developmental biology↗

bioRxiv2025

The AUUUC repeat RNA aggregates sequester RNA-binding proteins like NOVA2 and lead to iron dyshomeostasis in spinocerebellar ataxia type 37

Transcribed nucleotide repeat expansions can contribute to disease by altering RNA structure and function. Spinocerebellar ataxia type 37 (SCA37) is a neurodegenerative disorder caused by a pathogenic ATTTC repeat insertion within a non-pathogenic ATTTT repeat in the 5 untranslated region of DAB1. We have shown that the AUUUC repeat RNA forms aberrant nuclear aggregates in cells, subsequently confirmed by others in neurons from subjects with familial adult myoclonic epilepsy carrying a similar ATTTC repeat insertion. However, the mechanism by which these RNA aggregates cause neuropathology remains unknown. Here, we show that overexpression of the ATTTC repeat in human neural stem cells leads to the formation of abnormal nuclear RNA aggregates, supporting an AUUUC repeat-mediated mechanism of pathology through the sequestration of RNA-binding proteins (RBP). We identified 12 AUUUC repeat-interacting RBPs with specific neuronal functions, including NOVA2, which we demonstrate to colocalize with the AUUUC repeat aggregates. Moreover, we further investigated the accumulation of iron in these aggregates and observed a significant colocalization of iron and NOVA2 hotspots in ATTTC repeat-expressing cells, a pattern absent in control cells. Together, these findings uncover a novel RNA-mediated mechanism of pathology involving both RBPs and iron, expanding the current understanding of RNA repeat toxicity.

cell biology↗

bioRxiv2025

Embryonic Spinocerebellar Ataxia Type 37 AUUUC Repeat RNA Causes Neurodevelopmental Defects in Zebrafish

Onset of many neurodegenerative and neuromuscular diseases usually starts in adulthood; however, recent advances point toward neurodevelopmental changes as drivers of late neurodegeneration. How early neuropathological features occur in these conditions remains unclear, which is critical for timely therapeutic intervention. Here, we provide evidence that neurodevelopmental axonal defects initiate a motor phenotype in a zebrafish model of spinocerebellar ataxia type 37 (SCA37), a degenerative hereditary condition caused by an ATTTC repeat in the DAB1 gene. We investigated neuronal defects triggered by the embryonic AUUUC repeat RNA and their effects later in life by transiently expressing this RNA in embryos and analyzing innervation and motor function. We found abnormalities in motor neuron axonal outgrowth and muscle innervation. We also discovered disrupted embryonic motor activity and reduced locomotor distance and velocity in late adult zebrafish, demonstrating motor impairment. Moreover, we showed that NOVA2 expression rescues axonal defects, indicating dysfunction of NOVA2-regulated neurodevelopmental processes. Overall, our results establish embryonic expression of the AUUUC repeat RNA as a driver of axonal and synaptic abnormalities, interfering with neuronal circuits and culminating in adult motor dysfunction.

neuroscience↗

bioRxiv2025

The Insertion of an ATTTC Repeat in an Alu Element Hyperactivates a Primate-Specific Neurodevelopmental Enhancer in Spinocerebellar Ataxia Type 37

Alu are evolutionarily very old primate-specific interspersed repeat elements that constitute [~]11% of the human genome. They are a source of short tandem repeats (STRs), which often expand in size and originate inherited neuromuscular and neurodegenerative disorders. How expanded STR insertion mutations within Alu STRs culminate in disease remains unknown. Here we report an Alu STR located in an intron of DAB1 that functions as a neurodevelopmental enhancer. We demonstrated that an ATTTC repeat insertion in this DAB1 Alu STR, known to cause spinocerebellar ataxia type 37 (SCA37), hyperactivates a neurodevelopmental DAB1 enhancer. Importantly, we showed that neurons derived from SCA37 subjects have higher levels of DAB1 expression and DAB1 overexpression causes abnormal axonal pathfinding in vivo. Overall, these results establish that neuronal dysregulation of a developmental DAB1 Alu STR enhancer contributes to SCA37 pathogenesis, an unexplored mechanism likely acting in many Alu STR diseases, potentially reshaping the therapeutic landscape.

genetics↗

bioRxiv2024

The plant pioneer factor LFY uses trans-kingdom chromatin remodeling mechanisms in vertebrates

Multicellular complexity is thought to have evolved independently in animals and plants, but the molecular constraints driving this complexity are poorly understood. Genetic networks activated during animal and plant development both involve the regulation of nucleosome occupancy by chromatin remodelers. Pioneer factors, often specific to plants or animals, bind to nucleosome-occupied DNA in a sequence-specific manner, recruiting chromatin remodelers to control lineage-specific genetic networks. In this study, a plant-specific pioneer factor, LFY, when expressed in zebrafish cells, recruits the vertebrate chromatin remodeler Smarca4a to make DNA accessible at LFY binding sites. This allows a plant LFY-controlled promoter, introduced into the zebrafish genome, to become transcriptionally active in a Smarca4a-dependent manner. Our findings suggest that the protein-protein interaction between pioneer factors and chromatin remodelers is a trans-kingdom mechanism likely reflecting ancient eukaryotic events that became a key evolutionary constraint in the path to biological complexity.

evolutionary biology↗

bioRxiv2024

PERK inhibition in zebrafish mimics human Wolcott-Rallison syndrome phenotypes

BackgroundPERK (EIF2AK3) is an endoplasmic reticulum stress kinase whose loss of function disturbs human development, leading to skeletal dysplasia and permanent neonatal diabetes, as observed in the Wolcott-Rallison Syndrome (WRS). The lack of effective, less invasive therapies for developmental diseases highlights the need for animal models that replicate complex pathological phenotypes, while allowing scalable drug screening. Zebrafish, with their high fecundity and rapid development, facilitate efficient in vivo drug testing. MethodsWe aimed to assess the potential of zebrafish for studying PERK function and its pharmacological modulation, particularly as a model for developmental diseases like WRS. Bioinformatic analyses assessed the similarity between human and zebrafish PERK. Increasing concentrations of GSK2606414 were used to inhibit PERK. A combination of behavioural and functional assays evaluated the effects of GSK2606414 on zebrafish skeletal, neuromuscular, and cardiac development. Fluorescence microscopy in transgenic zebrafish expressing fluorescent pancreatic markers and a glucose probe assessed the diabetic-like phenotype. ResultsWe found high similarity between human and zebrafish PERK, along with bioactivity of the PERK inhibitor GSK2606414 in zebrafish. PERK inhibition evoked defects in WRS relevant parameters, such as growth and skeletal development, as well as neuromuscular and cardiac deficiencies, whereas parameters not associated with WRS like otolith area and eye/body ratio remained unaffected. Moreover, PERK inhibition decreased pancreatic ! cell mass and disrupted glucose homeostasis, indicating a diabetic phenotype. ConclusionThese findings evidence zebrafishs potential for studying PERK function and its pharmacological modulation in developmental disorders like WRS, aiding research on pathophysiology and experimental treatments.

pharmacology and toxicology↗

bioRxiv2023

Unravelling the regulatory function of a short 3'UTR sequence in zebrafish and in human cells

The mechanisms of gene expression regulation are essential for cell identity and function, and their disruption usually leads to human disease. The 3 untranslated region (UTR) of mRNA contains important regulatory elements of gene expression, including upstream sequence elements (USEs) that are cis-regulatory sequences localized upstream of polyA signals (PAS). One of the best functionally characterised USEs is located in the 3UTR of the Drosophilas polo gene, which disruption leads to critical phenotypes in adult flies. In this work we found that the USE of the Drosophilas polo gene (DplUSE) is also found in in the 3UTR of vertebrate genes, including zebrafish, mouse and human genes, showing higher levels of conservation than the whole 3UTR sequence. Using reporter assays, we show that DplUSE is able to increase gene expression in vitro in human cell lines and in vivo in zebrafish embryos. Importantly, in humans, the DplUSE containing genes are enriched for genes associated to serious diseases such as Congenital abnormalities and Malignant neoplasms, illustrating the potential of this sequence to modulate genes with relevant biological functions and related with human health. Concomitantly, when sequestering the molecular machinery that operates at the DplUSE using a dominant negative strategy, we show that this is enough to dysregulate DplUSE containing genes in human cells and disrupt proper embryo development in zebrafish. Aiming to understand the molecular mechanism operating at the DplUSE, we identified three RNA binding proteins (RBP) that specifically bind to the DplUSE in vertebrates. Importantly, one of such RBPs is PTBP1, the vertebrate orthologue of the fruit flys RBP Heph, that was demonstrated to be required for the DplUSE function in Drosophila. To test if PTBP1is essential for the DplUSE function, as observed in Drosophila, we depleted PTBP1 from human cells and observed a downregulation of the expression of DplUSE containing genes, demonstrating that the molecular mechanisms that operate at DplUSE are ultra-conserved. Finally, we explored if variants in DplUSE consensus could be associated to human disease. We found a reported single nucleotide polymorphism (SNP; rs3087967) that is associated with malignant tumor of colon and generates an ectopic consensus of DplUSE in the 3 UTR of the tumorigenic POU2AF2/C11orf53 gene. We further show that this ectopic DplUSE motif causes a gain-of-function in vivo in zebrafish gut cells, suggesting its involvement in colon cancer development. These results show that a short motif present in the 3UTR of genes from phylogenetically distant bilaterians, from fruit flies to humans, control genes expression through an ultra-conserved mechanism involving RBPs binding and its dysregulation might impact in human disease.

molecular biology↗

bioRxiv2023

FOXM1 expression reverts aging chromatin profiles through repression of the senescence-associated pioneer factor AP-1

Aging is characterized by changes in gene expression that drive deleterious cellular phenotypes leading to senescence. The transcriptional activation of senescence genes has been mainly attributed to epigenetic shifts, but the changes in chromatin accessibility and its underling mechanisms remain largely elusive in natural aging. Here, we profiled chromatin accessibility in human dermal fibroblasts (HDFs) from neonatal and octogenarian individuals. We found that AP-1 binding motifs are prevalent in elderly specific accessible regions of the chromatin while neonatal-specific regions are highly enriched for TEAD binding motifs. We further show that TEAD4 and FOXM1 share a conserved transcriptional regulatory landscape controlled by an age-dependent enhancer that closes with aging and drives senescence when deleted. Finally, we demonstrate that FOXM1 ectopic expression in elderly cells partially resets chromatin accessibility to a youthful state due to FOXM1 repressive function in the promoters of several members of the AP-1 complex. These results place FOXM1 at a top hierarchical level in chromatin remodeling required to prevent senescence.

genetics↗

bioRxiv2021

Theoretical causes of the Brazilian P.1 and P.2 lineages of the SARS-CoV-2 virus through molecular dynamics

The new {beta}-coronavirus has been causing sad losses around the world and the emergence of new variants has caused great concern. This pandemic is of a proportion not seen since the Spanish Flu in 1918. Thus, throughout this research, the B.1.1.28 lineage of the P.1 clade (K417T, N501Y, E484K) that emerged in Brazil was studied, as well as the latest Delta variant. This is because the molecular mechanisms by which phenotypic changes in transmissibility or mortality remain unknown. Through molecular dynamics simulations with the NAMD 3 algorithm in the 50ns interval, it was possible to understand the impact on structural stabilization on the interaction of the ACE2-RBD complex, as well as simulations in 30ns for the neutralizing antibody P2B-2F6, with this antibody was derived from immune cells from patients infected with SARS-CoV-2. Although not all molecular dynamics analyzes support the hypothesis of greater stability in the face of mutations, there was a predominance of low fluctuations. Thus, 3 (three) analyzes corroborate the hypothesis of greater ACE2-RBD stability as a result of P.1, among them: Low mean RMSF values, greater formation of hydrogen bonds and low solvent exposure measured by the SASA value. An inverse behavior occurs in the interaction with neutralizing antibodies, since the mutations induce greater instability and thus hinder the recognition of antibodies in neutralizing the Spike protein, where we noticed a smaller number of hydrogen bonds as a result of P.1. Through MM-PBSA energy decomposition, we found that Van der Waals interactions predominated and were more favorable when the structure has P.1 strain mutations. Therefore, we believe that greater stabilization of the ACE2-RBD complex may be a plausible explanation for why some mutations are converging in different strains, such as E484K and N501Y. The P.1 concern variant still makes the Spike protein recognizable by antibodies, and therefore, even if the vaccines efficacy can be diminished, there are no results in the literature that nullify them.

biophysics↗

bioRxiv2020

Leprosy in wild chimpanzees

Humans are considered the main host for Mycobacterium leprae, the aetiologic agent of leprosy, but spill-over to other mammals such as nine-banded armadillos and red squirrels occurs. Although naturally acquired leprosy has also been described in captive nonhuman primates, the exact origins of infection remain unclear. Here, we report on leprosy-like lesions in two wild populations of western chimpanzees (Pan troglodytes verus) in the Cantanhez National Park, Guinea-Bissau, and the Tai National Park, Cote dIvoire, West Africa. Longitudinal monitoring of both populations revealed the progression of disease symptoms compatible with advanced leprosy. Screening of faecal and necropsy samples confirmed the presence of M. leprae as the causative agent at each site and phylogenomic comparisons with other strains from humans and other animals show that the chimpanzee strains belong to different and rare genotypes (4N/O and 2F). The independent evolutionary origin of M. leprae in two geographically distant populations of wild chimpanzees, with no prolonged direct contact with humans, suggests multiple introductions of M. leprae from an unknown animal or environmental source.

evolutionary biology↗

bioRxiv2020

Cis-regulatory similarities in the zebrafish and human pancreas uncover potential disease-related enhancers

Introductory paragraphThe pancreas is a central organ for human diseases that have a dramatic societal burden, such as pancreatic cancer and diabetes1,2. Non-coding cis-regulatory elements (CREs) of DNA control gene expression3,4, being required for proper pancreas function. Most disease-associated alleles5,6 are non-coding, often overlapping with CREs5, suggesting that alterations in these regulatory sequences contribute to human pancreatic diseases by impairing gene expression. However, functional testing of CREs in vivo is not fully explored. Here we analysed histone modifications, transcription, chromatin accessibility and interactions, to identify zebrafish pancreas CREs and their human functional equivalents, uncovering disease-associated sequences across species. We found a human pancreatic enhancer whose deletion impairs the tumour suppressor gene ARID1A expression, conferring a potential tumour suppressor role to this non-coding sequence. Additionally, we identified a zebrafish ptf1a distal enhancer which deletion generates pancreatic agenesis, demonstrating the causality of this condition in humans7 and the interspecies functional equivalency of enhancers.

genomics↗