Biology subjects

Puig, S.

Publications and source records attributed to Puig, S..

16 recordsLinked to original sources

bioRxiv2026

Spatially resolved diversity in molecular states underlies congenital melanocytic nevi and associated tumors

The congenital melanocytic nevus (CMN) is a developmental skin disorder characterized by prenatal melanocyte overgrowth, exhibiting heterogeneity in surface size, depth, and clinical behavior. Large/giant CMN carry an elevated, anticipatory melanoma risk relative to more common, small CMN. However, deeper insights into melanocytic states, genomic and epigenomic alterations, and microenvironmental cues governing disease progression are needed to predict lesions disposed to transformation. Although large/giant CMN melanocyte heterogeneity was recently established, spatial organization of these states and their niche interactions are unknown. Using advanced spatial and single-cell transcriptomics and bulk methylomics, we characterized ten CMN, seven CMN-associated proliferative nodules and three clinically diagnosed melanomas arising in CMN from children, integrating data from healthy skin references to contextualize melanocyte states in situ. CMN-specific melanocytic states, distinct in differentiation and proliferation, were spatially stratified, with immature melanocytes in deep dermis and more differentiated melanocytes approaching the epidermis. We then constructed a robust atlas of CMN cellular states by integrating single-cell transcriptomic data from five new large/giant CMN with published datasets, using it to deconvolute the spatial information. Cell-cell communication inference uncovered enhanced signaling (e.g. pleiotrophin, IGF1, periostin, semaphorin pathways) between CMN melanocytes, fibroblasts, and hair follicle-associated cells. Analysis of CMN-derived tumors, including longitudinal cases with [≥]2 samples, revealed divergent spatial melanocytic transcription distinguishing immune-enriched lesions from tumors with oncogenic/pro-invasive signatures. Collectively, these findings establish a spatially resolved framework linking melanocyte heterogeneity, signaling, and genomic instability in CMN, providing mechanistic insights to refine risk stratification and prognosis for CMN-associated tumors.

genetics↗

bioRxiv2026

Prioritizing Neuroactive Ligands Using Motif-Guided Virtual Discovery and Zebrafish Profiling

Virtual screening of ultra-large chemical libraries is a highly effective strategy for early-stage drug discovery. However, these pipelines often yield thousands of molecules that pass computational filters, and in silico-derived interaction energies do not consistently predict experimental efficacy. Furthermore, many high-affinity hits do not necessarily function effectively in an organism with tissues, barriers, and extensive off-target possibilities. A major hurdle in drug discovery is the prioritization of top candidates for rodent testing. Here, we introduce Rosetta Engine for Anchoring Ligands with a Motif ("REAL-M"), a novel computational screening algorithm that uses structural interaction data from the Protein Data Bank (PDB) to guide ligand placement and selection. Using the hypocretin receptor as a test case for this computational pipeline, 28 of 30 predicted antagonists significantly blocked binding of the cognate peptide agonist in a PRESTO-Tango cell-based reporter assay, including six chemically diverse molecules with comparable efficacy to preexisting antagonists. Three of the six molecules significantly mitigated hypocretin-induced larval zebrafish hyperactivity. Secondary testing with a zebrafish hcrtr2 null mutant ensured that behavioral phenotypes were not due to off-target interactions, which we did observe with preexisting antagonists. This pipeline is readily adaptable to the thousands of zebrafish proteins with highly conserved binding pockets.

pharmacology and toxicology↗

bioRxiv2025

Transcriptomic meta-analysis in plaque psoriasis: an integrative bioinformatic approach to deciphering the genetic landscape and molecular pathways

Psoriasis is a chronic inflammatory skin disease influenced by both genetic and environmental factors. Despite extensive research, its precise etiology remains unclear, posing significant challenges to understanding and treatment. The disease pathogenesis involves self-reactive T cells and immune-related cytokines. Genome-wide association studies have identified various susceptibility loci for immune-related diseases, but the underlying mechanisms remain only partially understood. Recent discoveries of critical signaling pathways, biological processes, and immune cell involvement have expanded our knowledge and offer hope for improved therapeutic strategies. This study aimed to enhance our understanding of psoriasis and proposes novel therapeutic approaches by employing integrated bioinformatics to identify signaling pathways and biological processes as potential disease markers. Presenting a systematic review and taking a meta-analytical approach to transcriptomic profiles, this investigation examined differential gene expression patterns across 44 studies involving 975 samples comparing lesional psoriasis, non-lesional psoriasis, and healthy controls. Consensus transcriptome signatures revealed a significant association between immune-related genes and psoriasis pathogenesis. Functional enrichment analysis identified several enriched pathways related to immunity and immune system processes. Comparison of these findings with the existing literature indicated that some immune-related genes were already known, while others are novel in the context of psoriasis. Additionally, novel gene analysis demonstrated psoriasis involvement in pathways such as gluconeogenesis, the FoxO signaling pathway, and mitophagy. This integrative approach confirmed classic genetic associations while uncovering novel gene expression patterns and pathways relevant to psoriasis. Notably, the disruption of the gluconeogenesis pathway emerged as a critical finding. These insights enhance our understanding of psoriasis pathophysiology and pave the way for targeted therapies, offering improved management options for affected individuals.

molecular biology↗

bioRxiv2025

Brain-wide mapping reveals temporal and sexually dimorphic opioid actions

While the molecular and cellular effects of opioids have been extensively studied, the precise mechanisms by which these drugs target specific brain regions over time remain unclear. Similarly, despite well-documented sex differences in opioid responses, the anatomical basis for this sexual dimorphism is not well characterized. To address these questions, we developed an automated, scalable, and unbiased approach for whole-brain anatomical mapping of the neuronal activity marker c-Fos in response to acute morphine exposure. Using ribbon scanning confocal microscopy, we imaged whole cleared brains from male and female wild-type mice at 1 hour and 4 hours post-morphine administration. Our whole-brain analysis of c-Fos expression revealed distinct patterns of morphine-induced regional brain activation across time and sex. Notably, we observed a greater number of structures with significant activity differences at 4 hours compared to 1 hour. In male mice, significant changes were primarily localized to regions within the dopamine system, whereas in female mice, they were concentrated in cortical regions. By combining high-throughput imaging with whole-brain expression analysis, particularly in the context of opioid actions, our approach provides a more comprehensive understanding of how drugs of abuse affect the brain.

neuroscience↗

bioRxiv2024

Regulation of Fentanyl Reward in Male and Female Mice by the Circadian Transcription Factor NPAS2

Synthetic opioids like fentanyl are highly potent and prevalent in the illicit drug market, leading to tolerance, dependence, and opioid use disorder (OUD). Chronic opioid use disrupts sleep and circadian rhythms, which persist even during treatment and abstinence, increasing the risk of relapse. The bodys molecular clock, regulated by transcriptional and translational feedback loops, controls various physiological processes, including the expression of endogenous opioids and their receptors. The circadian transcription factor NPAS2, highly expressed in the nucleus accumbens, may have a crucial function in opioid-related behaviors. Our study found sex-specific roles for NPAS2-mediated reward behaviors in male and female mice, including in fentanyl seeking and craving. We also identified specific cell types and transcriptional targets in the nucleus accumbens of both mice and humans by which NPAS2 may mediate the impact of fentanyl on brain physiology and in opioid reward-related behaviors. Ultimately, our findings begin to uncover the mechanisms underlying circadian rhythm dysfunction and opioid addiction.

neuroscience↗

bioRxiv2024

Ribosome-Associated Vesicles promote activity-dependent local translation

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its deployment to local translation sites remain unclear. Here, we employed a multi-scale imaging approach combining cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify endoplasmic reticulum-derived Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes and promoting activity-dependent local translation. We demonstrate that neuronal stimulation triggers compartment-specific RAV responses: dendrites accumulate stationary RAVs at sites of enhanced translation, while axons accelerate RAV transport. Real-time imaging of translation at single mRNA resolution reveals that RAVs boost local translation output compared to RAV-independent mechanisms. These findings establish RAVs as specialized platforms that integrate activity-dependent signals with local protein synthesis, providing a mechanistic framework for understanding how neurons achieve precise spatiotemporal control of protein synthesis.

neuroscience↗

bioRxiv2024

Peripheral opioid tolerance involves skin keratinocytes and platelet-derived growth factor type B signaling

Opioid analgesic tolerance drives dose escalation which hampers the therapeutic utility of opioids by increasing centrally mediated deleterious side-effects, including respiratory depression or addiction. Peripheral opioid delivery provides a safer, effective alternative to systemic delivery by avoiding centrally mediated opioid side-effects. However, tolerance still occurs peripherally via mechanisms that remain unknown. Centrally, activation of the mu-opioid receptor (MOPr) by opioids induces release of platelet-derived growth factor-B (PDGF-B); and inhibition of PDGF receptor beta (PDGFR{beta}) prevents opioid tolerance. In the periphery, MOPr and PDGF-B are expressed in keratinocytes, and PDGFR{beta} is expressed in peripheral sensory neurons (PSNs), which are involved in tolerance. Previous studies showed that optogenetic stimulation of keratinocytes modulates PSNs via release of keratinocyte-derived factors. Thus, we hypothesized that mechanisms of peripheral opioid tolerance involve keratinocytes and PDGFR{beta} signaling. Using behavioral pharmacology, optogenetics and imaging in mice, we found that selective inhibition of peripheral PDGFR{beta} prevents peripheral morphine tolerance caused by repeated intraplantar (i.pl.) morphine injections. In addition, we show that PDGF-B is both necessary and sufficient to cause peripheral morphine tolerance. Repeated peripheral morphine injections lead to an increase in PDGF-B mRNA in MOPr-expressing keratinocytes and induce changes in the biophysical properties of keratinocytes as measured by patch-clamp electrophysiology. In parallel, we discovered that repeated optogenetic activation of keratinocytes is sufficient to induce peripheral morphine tolerance in a PDGF-B/PDGFR{beta}-dependent manner. Together, we show a novel epithelial-neuronal communication mechanism that incorporates keratinocytes and PDGF-B/PDGFR{beta} to mediate peripheral opioid tolerance, opening the door to safer, more effective pain therapeutics. Significance StatementPeripheral opioids are a safer alternative to systemic opioids. However, peripheral tolerance leads to reduction of analgesia over time, hampering clinical use of peripheral opioids. Here, we highlight a novel epithelial-neuronal communication mechanism that mediates peripheral tolerance. We discovered that intraplantar (i.pl.) morphine injections in mice cause peripheral tolerance via release of platelet-derived growth factor type B (PDGF-B) and activation of platelet-derived growth factor beta (PDGFR{beta}). We find that morphine i.pl. increases PDGF-B in mu-opioid receptor-expressing keratinocytes, which could be released to activate PDGFR{beta} in cutaneous nociceptor endings to mediate peripheral tolerance. Moreover, we show that photostimulation of keratinocytes is sufficient to cause peripheral tolerance in a PDGF-B/PDGFR{beta}-manner. Thus, keratinocytes and PDGF-B are new promising targets for peripheral opioid tolerance.

neuroscience↗

bioRxiv2023

Three-dimensional characterization of medium spiny neuron heterogeneity in the adult mouse striatum

Striatal projection neurons (SPNs) are traditionally segregated into two subpopulations expressing dopamine (DA) D1-like or D2-like receptors. However, this dichotomy is challenged by recent evidence. Functional and expression studies raise important questions: do SPNs co-express different DA receptors, and do these differences reflect unique striatal spatial distributions and expression profiles? Using RNAscope in mouse striatum, we report heterogenous SPN subpopulations distributed across dorsal-ventral and rostral-caudal axes. SPN subpopulations co-express multiple DA receptors, including D1 and D2 (D1/2R) and D1 and D3. Our integrative approach using single-nuclei multi-omics analyses provides a simple consensus to describe SPNs across diverse datasets, connecting it to complementary spatial mapping. Combining RNAscope and multi-omics shows D1/2R SPNs further separate into distinct subtypes according to spatial organization and conserved marker genes. Each SPN cell type contributes uniquely to genetic risk for neuropsychiatric diseases. Our results bridge anatomy and transcriptomics to offer new understandings of striatal neuron heterogeneity.

neuroscience↗

bioRxiv2023

Uncovering circadian rhythm disruptions of synaptic proteome signaling in prefrontal cortex and nucleus accumbens associated with opioid use disorder

Opioid craving and relapse vulnerability is associated with severe and persistent sleep and circadian rhythm disruptions. Understanding the neurobiological underpinnings of circadian rhythms and opioid use disorder (OUD) may prove valuable for developing new treatments for opioid addiction. Previous work indicated molecular rhythm disruptions in the human brain associated with OUD, highlighting synaptic alterations in the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc)--key brain regions involved in cognition and reward, and heavily implicated in the pathophysiology of OUD. To provide further insights into the synaptic alterations in OUD, we used mass-spectrometry based proteomics to deeply profile protein expression alterations in bulk tissue and synaptosome preparations from DLPFC and NAc of unaffected and OUD subjects. We identified 55 differentially expressed (DE) proteins in DLPFC homogenates, and 44 DE proteins in NAc homogenates, between unaffected and OUD subjects. In synaptosomes, we identified 161 and 56 DE proteins in DLPFC and NAc, respectively, of OUD subjects. By comparing homogenate and synaptosome protein expression, we identified proteins enriched specifically in synapses that were significantly altered in both DLPFC and NAc of OUD subjects. Across brain regions, synaptic protein alterations in OUD subjects were primarily identified in glutamate, GABA, and circadian rhythm signaling. Using time-of-death (TOD) analyses, where the TOD of each subject is used as a time-point across a 24- hour cycle, we were able to map circadian-related changes associated with OUD in synaptic proteomes related to vesicle-mediated transport and membrane trafficking in the NAc and platelet derived growth factor receptor beta signaling in DLPFC. Collectively, our findings lend further support for molecular rhythm disruptions in synaptic signaling in the human brain as a key factor in opioid addiction.

neuroscience↗

bioRxiv2022

A spatial atlas of Wnt and Frizzled receptor expression in adult mouse liver

Hepatic zonation is critical for most metabolic functions in liver. Wnt signaling plays an important role in establishing and maintaining liver zonation. Yet, the anatomic expression of Wnt signaling components, including all 10 Frizzled receptors (Fzds), has not been characterized in adult liver. To address this, we quantitatively mapped the spatial expression of Wnt/Fzd pathway components in adult mouse liver via multiplex fluorescent in situ hybridization. While all 10 Fzds are expressed within a metabolic unit, Fzds 1, 4, and 6 are the highest expressed. Though the majority of Wnt signaling occurs in zone 3, expression of most Fzds is not zonated. In contrast, Fzd6 is preferentially expressed in zone 1. We also discovered that Wnt2 and Wnt9b expression is highly zonated and primarily found in zone 3. Therefore, our results suggest that zonated Wnt expression is critical for zonation maintenance in healthy adult liver. Finally, we showed that Fzds and Wnts are not uniformly expressed by all hepatic cell types. Rather, there is broad distribution among both hepatocytes and non-parenchymal cells, including endothelial cells. Overall, our establishment of a definitive mRNA expression atlas of Wnt/Fzd pathway components opens the door to future functional characterization in healthy and disease states.

cell biology↗

bioRxiv2022

Gene expression and protein synthesis remodeling in response to iron deficiency in yeast

Iron is an essential trace element that serves as a cofactor for enzymes involved in multiple metabolic pathways, including ribosome biogenesis, protein translation, DNA synthesis and repair, lipid metabolism, and mitochondrial oxidative phosphorylation. In eukaryotes, iron deficiency leads to global inhibition of protein synthesis and coordinated changes in gene expression to limit iron utilization. Although several steps of protein translation depend on iron-containing enzymes, the contribution of iron to the translation process is not understood at the molecular level. Here, we report a genome-wide analysis of protein translation in response to iron deficiency in yeast using ribosome profiling. We show that iron depletion affects global protein synthesis as well as leads to translational repression of several groups of genes involved in iron-related processes. We further demonstrate that the RNA-binding proteins Cth1 and Cth2 play a central role in controlling the changes in protein translation by repressing the activity of the iron-dependent Rli1 ribosome recycling factor, inhibiting mitochondrial translation, and affecting the translation of genes involved in heme biosynthesis. We also discovered a mechanism, whereby iron deficiency represses translation of MRS3 mRNA, encoding mitochondrial iron transporter, through increased expression of antisense long non-coding RNA. Together, our results reveal complex gene expression and protein synthesis remodeling in response to low iron showing how this important metal affects protein translation at multiple levels.

genetics↗

bioRxiv2022

A role for the circadian transcription factor NPAS2 in the progressive loss of non-rapid eye movement sleep and increased arousal during fentanyl withdrawal in male mice

AbstractO_ST_ABSRationaleC_ST_ABSSynthetic opioids like fentanyl are contributing to the rise in rates of opioid use disorder and drug overdose deaths. Sleep dysfunction and circadian rhythm disruption may worsen during opioid withdrawal and persist during abstinence. Severe and persistent sleep and circadian alterations are putative factors in opioid craving and relapse. However, very little is known about the impact of fentanyl on sleep architecture and sleep-wake cycles, particularly opioid withdrawal. Further, circadian rhythms regulate sleep-wake cycles, and the circadian transcription factor, neuronal PAS domain 2 (NPAS2) is involved in the modulation of sleep architecture and drug reward. Here, we investigate the role of NPAS2 in fentanyl-induced sleep alterations. ObjectivesTo determine the effect of fentanyl administration and withdrawal on sleep architecture, and the role of NPAS2 as a factor in fentanyl-induced sleep changes. MethodsElectroencephalography (EEG) and electromyography (EMG) was used to measure non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) at baseline and following acute and chronic fentanyl administration in wild-type and NPAS2-deficient male mice. ResultsAcute and chronic administration of fentanyl led to increased wake and arousal in both wild-type and NPAS2-deficient mice, an effect that was more pronounced in NPAS2-deficient mice. Chronic fentanyl administration led to decreased NREMS, which persisted during withdrawal, progressively decreasing from day 1 to 4 of withdrawal. The impact of fentanyl on NREMS and arousal was more pronounced in NPAS2-deficient mice. ConclusionsChronic fentanyl disrupts NREMS, leading to a progressive loss of NREMS during subsequent days of withdrawal. Loss of NPAS2 exacerbates the impact of fentanyl on sleep and wake, revealing a potential role for the circadian transcription factor in opioid-induced sleep changes.

neuroscience↗

bioRxiv2022

Sex-specific role of the circadian transcription factor NPAS2 in opioid tolerance, withdrawal, and analgesia

Opioids like fentanyl are the mainstay treatment for chronic pain. Unfortunately, opioids high addiction liability has led to the current opioid crisis. This is in part related to long-term use side-effects, including analgesic tolerance (gradual decrease in analgesia), and physical dependence (withdrawal symptoms upon opioid interruption), causing dose-escalation and preventing usage interruption. Altered circadian rhythmicity and sleep patterns are common in patients on opioid therapy. Prior research showed intricate bidirectional interactions between circadian rhythmicity, opioid analgesic efficacy, and side-effects. However, underlying mechanisms are largely unknown. Neuronal PAS domain protein 2 (NPAS2) is a circadian transcription factor that is highly expressed in structures of the central nervous system that modulate pain and opioids. In this study, we used male and female mice expressing non-functional NPAS2 (NPAS2 deficient, NPAS2-/-) to investigate the role of NPAS2 in fentanyl analgesia, tolerance, hyperalgesia, and dependence. In NPAS2-/- mice, we found that thermal pain thresholds, acute analgesia, and tolerance to a fixed dose of fentanyl were largely like wild-type mice. However, female NPAS2-/- mice augmented behavioral state of analgesic tolerance and exhibited significantly more behavioral symptoms of physical dependence. Conversely, only male NPAS2-/- mice had increased fentanyl-induced hypersensitivity, when compared to matched-sex littermate controls. Together, our findings suggest sex-specific effects of NPAS2 signaling in the regulation of fentanyl-induced tolerance, hyperalgesia, and dependence.

neuroscience↗

bioRxiv2022

Deficiency of the RNA-binding protein Cth2 extends yeast replicative lifespan by alleviating its repressive effects on mitochondrial function

Iron dyshomeostasis contributes to aging, but little information is available about the molecular mechanisms. Here, we provide evidence that, in Saccharomyces cerevisiae, aging is associated with altered expression of genes involved in iron homeostasis. We further demonstrate that defects in the conserved mRNA-binding protein Cth2, which controls stability and translation of mRNAs encoding iron-containing proteins, increase lifespan by alleviating its repressive effects on mitochondrial function. Mutation of the conserved cysteine residue in Cth2 that inhibits its RNA-binding activity is sufficient to confer longevity, whereas Cth2 gain-of-function shortens replicative lifespan. Consistent with its function in RNA degradation, we demonstrate that Cth2 deficiency relieves Cth2-mediated post-transcriptional repression of nuclear-encoded components of the electron transport chain. Our findings uncover a major role of the RNA-binding protein Cth2 in the regulation of lifespan and suggest that modulation of iron starvation signaling can serve as a target for potential aging interventions.

genomics↗

bioRxiv2020

Lateralized decrease of parvalbumin+ cells in the somatosensory cortex of ASD models is correlatedwith unilateral tactile hypersensitivity

Inhibitory control of excitatory networks contributes to cortical functions. Increasing evidence indicates that parvalbumin expressing (PV+) basket cells (BC) are a major player in maintaining the balance between excitation (E) and inhibition (I) in the cortex. Disruption of E/I balance in cortical networks is believed to be a hallmark of autism spectrum disorders (ASD) and may contribute to sensory alterations seen in ASD. Here, we report a lateralized decrease in the number of PV+ BCs in L2/3 of the somatosensory cortex in the dominant hemisphere of adult Shank3-/- and Cntnap2-/- mouse models of ASD. The dominant hemisphere was identified during a reaching task to establish each animals dominant forepaw. Double labeling with anti-PV antibody and a biotinylated lectin (i.e., VVA) showed that the number of BCs was not different but rather, some BCs did not express detectable levels of PV (PV-), resulting in an elevated number of PV- VVA+ basket cells. This lateralized reduction was not observed in the number of interneurons from the other two major groups that express somatostatin or the serotonergic receptor 5HT3a. Finally, we showed that dominant hind paws had higher mechanical sensitivity (i.e., lower mechanical thresholds measured with von Frey test) but no difference in thermal sensitivity (measured with Hargreaves test) when compared to the other hind paw. This mechanical hypersensitivity in the dominant paw correlated with the decrease in the number of PV+ interneurons and reduced PV expression in the corresponding cortex. Together, these results suggest that the sensory hypersensitivity in ASD could be due to decreased inhibitory inputs to the dominant somatosensory cortex.

neuroscience↗

bioRxiv2019

Inherited duplications of PPP2R3B promote naevi and melanoma via a novel C21orf91-driven proliferative phenotype

The majority of the heredity of melanoma remains unexplained, however inherited copy number changes have not yet been systematically studied. The genetic environment is highly relevant to treatment stratification, and new gene discovery is therefore desirable. Using an unbiased whole genome screening approach for copy number we identify here a novel melanoma predisposing factor, familial duplications of gene PPP2R3B, encoding a regulatory unit of critical phosphatase PP2A. Significant correlation between expression of PPP2R3B in tumour tissue and survival in a large melanoma cohort was confirmed, and associated with a non-immunological expression profile. Mechanistically, construction and extensive characterization of a stable, inducible cellular model for PPP2R3B overexpression revealed induction of pigment cell switching towards proliferation and away from migration. Importantly, this was independent of the known microphthalmia-associated transcription factor (MITF)-controlled pigment cell phenotype switch, and was instead driven by uncharacterised gene C21orf91. Bioinformatic studies point to C21orf91as a novel target of MITF, and therefore a potential hub in the control of phenotype switching in melanoma. This study identifies novel germline copy number variants in PPP2R3B predisposing to melanocytic neoplasia, and uncovers a new potential therapeutic target C21orf91 in the control of pigment cell proliferation.

genomics↗