Biology subjects

Sampaio, P.

Publications and source records attributed to Sampaio, P..

7 recordsLinked to original sources

bioRxiv2026

Dual Ligand Cooperation at the Plasma Membrane Drives Transport of Engineered Small Extracellular Vesicles Across Brain Endothelial Cells

The natural delivery properties of small extracellular vesicles (sEVs) can be harnessed and enhanced through engineering to create a new class of biotherapeutics, particularly for central nervous system (CNS) disorders. While evidence supports the ability of sEVs to cross biological barriers and deliver functional cargo to target cells, a limited understanding of their uptake and transport across the brain hinders their translational potential. In this study, we investigated either native and engineered sEVs, developed by us, using a novel modular engineering platform that employs a dual-targeting strategy to facilitate uptake and transport through human brain endothelial cells (BECs). By utilizing super-resolution microscopy, we provided direct insights into the mechanisms of docking, intracellular sorting, and transport of engineered sEVs. The engineered sEVs formulation demonstrated significantly enhanced uptake, intracellular trafficking across BECs, and the ability to bypass degradative pathways. In vivo, the engineered sEVs exhibited preferential accumulation in the brain choroid plexus, a structure located within the lateral and fourth ventricles, thereby effectively targeting the blood-cerebrospinal fluid (CSF) barrier. These findings highlight the potential of combining advanced targeting strategies with high-resolution imaging to study sEV interactions with the brain biological barriers and develop more effective CNS therapies.

bioengineering↗

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↗

bioRxiv2023

Characterization of peroxidasin expression in histologically normal human adult and fetal kidney tissue

Peroxidasin (PXDN) is an enzyme of the peroxidase family that plays a critical role in extracellular matrix (ECM) formation and tissue development. In the kidney, very few studies using animal cells suggested that PXDN may have a role in the maintenance of the structural and functional integrity of the renal ECM. Still, the normal expression and (patho)physiological roles of PDXN in the mature and developing human kidney remain unknown. In this work we used fluorescent immunohistochemistry and advanced microscopy and image processing methodologies to perform a quantitative characterization of PXDN expression in the different anatomic compartments of histologically normal kidney tissue, obtained from adult nephrectomy specimens, and from postmortem fetal and neonatal kidney specimens (excluding death by kidney disease and genitourinary developmental anomalies). Results show that PXDN is expressed in all mature kidney compartments, but at significantly higher levels in tubular epithelial cells, particularly in the distal tubules. A similar PXDN expression pattern was observed in the developing kidney, as early as the in the comma-shaped-body stage. In the fetal kidney, PXDN was diffusely expressed in the branches of the ureteric bud but not in the cells of the metanephric blastema. This is the first demonstration that PXDN is differentially expressed in histologically normal human renal parenchyma, exhibiting a remarkably consistent pattern of predominant tubular expression since the early stages of metanephrogenesis This data suggests a relevant, compartment-specific, role of PXDN in nephrogenesis and in the kidney physiology, highlighting a main role in tubular functions, that is worth further investigation.

physiology↗

bioRxiv2022

Fluid manipulations uncover mechanosensation for zebrafish left-right establishment during one-hour time interval

Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development (Shiratori and Hamada, 2006). The mechanism behind left-right establishment is not fully understood (Mizuno et al., 2020; Maerker et al., 2021; Minegishi et al., 2021). Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a decisive developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chemical determinants. Our findings revealed an unprecedented recovery capacity of the embryo to re-fil and re-circulate new LRO fluid. The embryos that later developed laterality problems were found to be those that had lower anterior angular velocity and thus less anterior-posterior heterogeneity. Aiming to test if this led to differential endocytosis of any molecular determinant, we replaced the LRO fluid by a physiological buffer. Despite some transitory flow homogenization, laterality defects were absent unless viscosity was altered demonstrating that symmetry breaking does not depend on the nature of the fluid content but rather relies on fluid mechanics. Altogether, we conclude that mechanosensing is the likely mechanism to govern left-right early establishment.

developmental biology↗

bioRxiv2018

Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast

Resolution in microscopy is not limited by diffraction as long as a nonlinear sample response is exploited. In a paradigmatic example, stimulated-emission depletion (STED) fluorescence microscopy fundamentally breaks the diffraction limit by using a structured optical pattern to saturate depletion on a previously excited sample area. Two-dimensional (2D) STED, the canonical low-noise STED mode, structures the STED beam by using a vortex phase mask, achieving a significant lateral resolution improvement over confocal fluorescence microscopy. However, axial resolution and optical sectioning remain bound to diffraction. Here we use a tunable coherent-hybrid (CH) beam to improve optical sectioning, markedly reducing background fluorescence. CH-STED, which inherits the 2D-STED immunity to spherical aberration, diversifies the depletion strategy, allowing an optimal balance between two key metrics (lateral resolution and background suppression) to be found. CH-STED is used to perform high-contrast imaging of complex biological structures, such as the mitotic spindle and the neuron cell body.

biophysics↗