Biology subjects

Almeida, R. G.

Publications and source records attributed to Almeida, R. G..

10 recordsLinked to original sources

bioRxiv2026

Live-imaging of endogenous neurofascins reveals glial adhesion shapes developing nodes of Ranvier

The organisation of myelinated axons into specialised domains is essential for saltatory conduction and depends on the polarised distribution of neuronal and glial cell-adhesion molecules, including neurofascins. However, how these domains assemble and refine in vivo remains unclear, as longitudinal imaging of endogenous proteins within intact nervous systems remains challenging. Here, we generated knock-in zebrafish in which endogenous neuronal and glial neurofascins are fused to fluorescent proteins, which enabled live-imaging without perturbing node assembly or introducing overexpression artefacts. Using these reporters, we visualised neuronal and glial neurofascin dynamics, and the formation of nodes of Ranvier and paranodes in vivo. We find that nascent nodes progressively compact in developing peripheral nerves, and that this process depends on glial neurofascin. Our novel toolkit for imaging endogenous neurofascins reveals a glia-dependent mechanism that shapes the developmental refinement of nodal architecture after their initial assembly. Our findings imply that glia-driven modulation of paranodes can be a mechanism for nervous systems to regulate circuit function.

neuroscience↗

bioRxiv2026

Discovery of Semicarbazone and Thiosemicarbazone Analogs as Competitive SARS-CoV-2 Virus Main Protease (Mpro) Inhibitors

Despite the development of vaccines and antivirals, coronavirus disease 2019 (COVID-19) continues to affect populations worldwide. Given the high mutation rate of the SARS-CoV-2 virus and reports of drug resistance, there is a continued need for new therapeutic options. SARS-CoV-2 main protease (Mpro) is essential for viral replication and is a conserved target among coronaviruses. Most known Mpro inhibitors target the active site, although allosteric sites have already been identified. In this study, we conducted a virtual screening of 2,060 compounds targeting an allosteric site of SARS-CoV-2 Mpro. From this screen, 41 computational hits and analogs were selected and evaluated using biochemical assays against SARS-CoV-2 Mpro. Among them, compound 25, a semicarbazone, demonstrated a half-maximal inhibitory concentration (IC50) of 99 M. Additionally, two thiosemicarbazone analogs (compounds 50 and 51) inhibited SARS-CoV-2 Mpro with IC50 values of 61 M and 70 M. Biochemical assays suggest that these compounds act as noncovalent competitive inhibitors of SARS-CoV-2 Mpro. Molecular dynamics simulations revealed that compound 25 is unstable at the allosteric site of SARS-CoV-2 Mpro but forms stable and favorable interactions at the active site, supporting its potential as a competitive inhibitor, a finding subsequently confirmed by biochemical assays. Our structure-based computational and biochemical approach identified semicarbazone and thiosemicarbazone scaffolds as promising candidates for the development of reversible SARS-CoV-2 Mpro inhibitors.

biochemistry↗

bioRxiv2025

Mislocalisation of TDP-43 to the cytoplasm of either neurons or oligodendrocytes causes axonopathy and dysmyelination

Neurons with large, myelinated axons are vulnerable to degeneration across the amyotrophic lateral sclerosis (ALS) frontotemporal dementia (FTD) disease spectrum. The defining molecular pathology of this spectrum is mislocalisation of the RNA binding protein TDP-43 from the nucleus to the cytoplasm. Even though cytoplasmic TDP-43 is prevalent in neurons and oligodendrocytes, how these molecular pathologies contribute to neurodegeneration remains unclear. Here we developed humanised zebrafish in which we restricted TDP-43 to the cytoplasm of either neurons or oligodendrocytes. Cytoplasmic TDP-43 restricted to neurons led to a severe axonopathy, with extreme distal axonal swelling and reduced myelination. Axonopathy was mirrored by cell type specific loss of TDP-43 function from neurons, pointing to loss of function, rather than a toxic gain of function of mislocalised TDP-43, as driving this phenotype. Preventing oligodendrocyte differentiation and myelination when TDP-43 was mislocalised in neurons exacerbated axonopathy, indicating that oligodendrocytes limit neurodegeneration. Indeed, when TDP-43 was mislocalised to the cytoplasm of oligodendrocytes this also led to axonopathy and reduced myelination, pointing to complex contributions of neurons and oligodendrocytes to neurodegeneration.

neuroscience↗

bioRxiv2024

Fast, Accurate, and Versatile Data Analysis Platform for the Quantification of Molecular Spatiotemporal Signals

Optical recording of intricate molecular dynamics is becoming an indispensable technique for biological studies, accelerated by the development of new or improved biosensors and microscopy technology. This creates major computational challenges to extract and quantify biologically meaningful spatiotemporal patterns embedded within complex and rich data sources, many of which cannot be captured with existing methods. Here, we introduce Activity Quantification and Analysis (AQuA2), a fast, accurate, and versatile data analysis platform built upon advanced machine learning techniques. It decomposes complex live imaging-based datasets into elementary signaling events, allowing accurate and unbiased quantification of molecular activities and identification of consensus functional units. We demonstrate applications across a wide range of biosensors, cell types, organs, animal models, and imaging modalities. As exemplar findings, we show how AQuA2 identified drug-dependent interactions between neurons and astroglia, and distinct sensorimotor signal propagation patterns in the mouse spinal cord.

neuroscience↗

bioRxiv2022

CNS myelination requires VAMP2/3-mediated membrane expansion in oligodendrocytes

Myelin is required for rapid nerve signaling and is emerging as a key driver of CNS plasticity and disease. How myelin is built and remodeled remains a fundamental question of neurobiology. Central to myelination is the ability of oligodendrocytes to add vast amounts of new cell membrane, expanding their surface areas by many thousand-fold. However, how oligodendrocytes add new membrane to build or remodel myelin is unknown. Here, we show that CNS myelin membrane addition requires exocytosis mediated by the vesicular SNARE proteins VAMP2/3. Genetic inactivation of VAMP2/3 in myelinating oligodendrocytes caused severe hypomyelination and premature death without overt loss of oligodendrocytes. Through live imaging, we discovered that VAMP2/3-mediated exocytosis drives membrane expansion within myelin sheaths to initiate wrapping and power sheath elongation. In conjunction with membrane expansion, mass spectrometry of oligodendrocyte surface proteins revealed that VAMP2/3 incorporates axon-myelin adhesion proteins that are collectively required to form nodes of Ranvier. Together, our results demonstrate that VAMP2/3-mediated membrane expansion in oligodendrocytes is indispensable for myelin formation, uncovering a cellular pathway that could sculpt myelination patterns in response to activity-dependent signals or be therapeutically targeted to promote regeneration in disease.

neuroscience↗

bioRxiv2022

A retroviral origin of vertebrate myelin

Myelin, the insulating sheath that surrounds neuronal axons, is produced by oligodendrocytes in the central nervous system (CNS). This evolutionary innovation, which first appears in jawed vertebrates, enabled rapid transmission of nerve impulses, more complex brains and greater morphological diversity. Here we report that RNA level expression of RNLTR12-int, a retrotransposon of retroviral origin, is essential for myelination. We show RNLTR12-int-encoded non-coding RNA binds to the transcription factor SOX10 to regulate transcription of myelin basic protein (Mbp, the major constituent of myelin) in rodents. RNLTR12-int like sequences (which we name RetroMyelin) are found in all jawed-vertebrates and we further demonstrate their function in regulating myelination in two different vertebrate phyla (zebrafish and frogs). Our study therefore suggests that retroviral endogenization was a key step in the emergence of vertebrate myelin.

neuroscience↗

bioRxiv2022

Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 {micro}M and 66 {micro}M. In addition, eight compounds inhibited PLpro with IC50 ranging from 1.7 {micro}M to 46 {micro}M. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

biochemistry↗

bioRxiv2021

Clusters of neuronal Neurofascin prefigure node of Ranvier position along single axons

The spacing of nodes of Ranvier crucially affects conduction properties along myelinated axons. It has been assumed that node position is primarily driven by the growth of myelin sheaths. Here, we reveal an additional mechanism of node positioning that is driven by the axon. We show through longitudinal live imaging of node formation dynamics that stable clusters of the cell adhesion molecule Neurofascin A accumulate at specific sites along axons prior to myelination. While some of these clusters change position upon encounter with growing myelin sheaths, others restrict sheath extension and are therefore predictive of future node position. Animals that lack full-length Neurofascin A showed increased internodal distances and less regular spacing of nodes along single axons. Together, our data reveal the existence of an axonal mechanism to position its nodes of Ranvier that does not depend on regulation of myelin sheath length.

neuroscience↗

bioRxiv2020

Synaptic vesicle fusion along axons is driven by myelination and subsequently accelerates sheath growth in an activity-regulated manner

To study activity-regulated myelination, we imaged synaptic vesicle fusion along single axons in living zebrafish, and found, surprisingly, that axonal synaptic vesicle fusion is driven by myelination. This myelin-induced axonal vesicle fusion was enriched along the unmyelinated domains into which newly-formed sheaths grew, and was promoted by neuronal activity, which in turn accelerated sheath growth. Our results indicate that neuronal activity consolidates sheath growth along axons already selected for myelination.

neuroscience↗

bioRxiv2019

Disruption to NKCC1 impairs the response of myelinating Schwann cells to neuronal activity and leads to severe peripheral nerve pathology

Myelinating Schwann cells of the peripheral nervous system (PNS) express numerous ion channels and transporters, and have the capacity to respond to neuronal activity. However, it remains unknown how the response of Schwann cells to neuronal activity affects peripheral nerve formation, health or function in vivo. Through a genetic screen in zebrafish, we identified a mutant, ue58, with severe disruption to the morphology of myelin along peripheral nerves and associated nerve oedema. Molecular analyses indicated that this phenotype was caused by the loss of function of a previously uncharacterized gene, slc12a2b, which encodes a zebrafish paralog of the solute carrier NKCC1. NKCC1 is a co-transporter of Na+, K+, and Cl- ions and water, typically from the extracellular space into cells. Upon impairing slc12a2b function, constitutively, or specifically in neurons or myelinating Schwann cells, we observed disruption to myelin and nerve oedema. Strikingly, we found that treatment of slc12a2b mutants with TTX completely prevented the emergence of these pathologies. Furthermore, TTX treatment rescued pathology in animals with cell-type specific loss of slc12a2b from myelinating Schwann cells. Together our data indicate that NKCC1 regulates ion homeostasis following neuronal activity and that this is required to maintain myelinated axon and peripheral nerve integrity.

neuroscience↗