Biology subjects

Santini, T.

Publications and source records attributed to Santini, T..

13 recordsLinked to original sources

bioRxiv2026

nHOTAIRM1 scaffolds ANXA11-dependent assemblies to drive axonal mRNA localization in human motor neurons

Long noncoding RNAs (lncRNAs) are well recognized as regulators of neuronal development and function, yet their integration into canonical axonal transport pathways, an essential component of spatial gene regulation in highly polarized cells, remains incompletely defined. It is still unclear to what extent individual lncRNAs confer cargo selectivity and couple discrete mRNA cohorts to established transport machineries. Here we identify a cytoplasmic role for the neuronal isoform of HOTAIRM1 (nHOTAIRM1) in shaping the axonal RNA landscape of human iPSC-derived spinal motor neurons (spMNs). We show that nHOTAIRM1 associates with Annexin A11 (ANXA11), a factor linked to lysosome-coupled movement of RNA granules and engages a defined subset of MN-relevant mRNAs. Transcriptome-wide and targeted interaction assays, together with computational mapping and steric competition, support direct RNA-RNA pairing between nHOTAIRM1 and mRNAs involved in cytoskeletal organization and synaptic or vesicular functions, while spatial analyses place these RNA pairs in close proximity within the soma and neurites. Loss-of-function experiments further show that nHOTAIRM1 depletion reduces the association of these mRNAs with ANXA11-positive complexes and diminishes their enrichment in neuronal projections, an effect that is recapitulated by ANXA11 depletion. Collectively, these findings support a model in which nHOTAIRM1 contributes to the selective recruitment of cargo mRNAs into ANXA11-associated transport assemblies, thereby promoting their localization within distal neurites. More broadly, our findings provide mechanistic insight into how a lncRNA can regulate mRNA sorting and compartment-specific RNA trafficking in human spMNs.

molecular biology↗

bioRxiv2026

An open-source stereotaxic container with an integrated cutting guide for human brain fixation during magnetic resonance imaging and sectioning for histology

IntroductionPostmortem imaging at ultrahigh field strengths, such as 7 Tesla (7T) magnetic resonance imaging (MRI), enables unprecedented visualization of fine brain structures and pathology. However, precise registration between MRI and histology is often compromised by tissue deformation and lack of standardized tools for preserving anatomical orientation. A reliable, high-resolution MRI-to-histology workflow is essential for MRI-guided sampling to investigate neurodegenerative diseases. MethodsWe developed a human postmortem brain container composed of stereotaxic enclosure and cutting guide system using iterative 3D printing. The final design is constructed from Victrex AM 200, a polyaryletherketone material engineered with MRI compatibility, high mechanical strength, and high resistance to fixatives. Human brains were embedded in an agar/sucrose mixture within the container and scanned at 7T using high-contrast and high-resolution imaging sequences. The cutting guide enables reproducible sectioning at defined tissue coronal slab thicknesses (9.2 {+/-} 0.6 mm). Block face photographs of each coronal slab were then co-registered with MRI for targeted tissue sampling. ResultsTo date, 215 human postmortem brains have been imaged ex vivo using this container. The container maintains consistent spatial orientation from imaging through sectioning, supporting correlation between MR-visible features and histological findings. Imaging-visible abnormalities, such as white matter hyperintensities and cerebral microbleeds, were localized and sampled with minimal deformation. The container enables single-scan workflows, avoiding custom cutting templates or repeat imaging. It supports deep learning-based lesion segmentation and MRI-guided histology registration, facilitating large-scale neuropathological investigations. ConclusionThe stereotaxic cutting guide system addresses a critical gap in postmortem imaging by integrating MRI-compatible materials, precision-guided sectioning, and high-throughput imaging. It has enabled multimodal studies of aging and neurodegeneration and is being adopted in neuropathological workflows. This platform provides a reproducible, scalable, and anatomically precise solution for aligning ultrahigh field MRI with histology, enhancing both clinical research and digital pathology efforts.

bioengineering↗

bioRxiv2026

LINC00205 acts as a multivalent scaffold promoting FUSP525L recruitment in Amyotrophic Lateral Sclerosis stress granules

Stress granules (SG) are dynamic, membrane-less ribonucleoprotein assemblies that orchestrate cellular stress responses and rapidly disassemble upon stress relief. In Amyotrophic Lateral Sclerosis (ALS), mutations in RNA-binding proteins such as Fused in Sarcoma (FUS) impair SG dynamics, promoting the formation of aberrant and persistent granules. Although long non-coding RNAs (lncRNAs) are emerging as regulators of ribonucleoprotein organization, their mechanistic role in SG architecture and pathological remodeling remains largely unexplored. Here, we identify LINC00205 as a critical RNA regulator of pathological SG dynamics in FUSP525L-associated ALS. Using Neuroblastoma cells and human iPSC-derived Motor Neurons (MN), we show that LINC00205 is enriched in SG upon oxidative stress and directly interacts with mutant FUSP525L. Knock-out of LINC00205 selectively reduces the formation of FUSP525L-containing SG and restores physiological SG disassembly kinetics, without affecting normal SG or FUS expression levels. Mechanistically, LINC00205 acts as a multivalent RNA scaffold, directly binding mRNAs specifically enriched in pathological SG, such as PLCXD3 and PIK3CA, as well as the RNA helicase DHX36, which is preferentially associated with FUSP525L-containing SG. We demonstrate that LINC00205 promotes their specific recruitment into pathological SG, thereby contributing to the aberrant features of these assemblies. Together, our findings uncover an active lncRNA-driven mechanism that shapes the molecular composition of aberrant ALS-related SG and regulates their persistence, establishing lncRNAs as key organizers of RNA-protein assemblies under stress and providing a conceptual framework for modulating pathological condensates.

molecular biology↗

bioRxiv2026

Neuronal late endosomes serve as selective RNA hubs disrupted by ALS-linked FUS mutation

Neurons depend on tightly regulated positioning of cellular components to maintain long-distance signaling, but the mechanisms guiding specific mRNAs to distant regions remain unclear. Here we show that late endosomes function as selective RNA carriers in human motor neurons and uncover the molecular logic guiding their loading. Using APEX2-mediated proximity labeling, we identify the external transcriptome of RAB7A-positive endosomes and find a specific population of mRNAs enriched for endosomal, axonal and synaptic functions. We find that mRNAs enriched in RAB7A endosomes contain evolutionarily conserved 5'UTRs which act as localization signals, and that the RNA-binding protein GEMIN5 interacts with these regions to promote endosomal RNA recruitment. Finally, we show that in ALS-associated conditions there is a conspicuous loss of endosome-associated transcripts and the mislocalization of GEMIN5 from endosomes. These findings uncover fundamental principles of RNA compartmentalization and highlight endosomal mRNA loading as a vulnerable axis in neuronal homeostasis.

molecular biology↗

bioRxiv2025

Mutant FUS perturbs m6A regulation by repressing ALKBH5, while restoring m6A levels alleviates ALS pathology in vivo

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder marked by the progressive loss of motor neurons, where aberrant stress granule (SG) dynamics has emerged as a hallmark, especially in cases involving mutations in RNA-binding proteins such as FUS and TDP-43. We previously showed that m6A RNA modification is elevated in FUS-associated ALS and that restoring its physiological levels through METTL3 inhibition is sufficient to rescue the aberrant stress granule phenotypes in different human cellular models. Importantly, here we show that reducing m6A levels ameliorates locomotor defects and neuromuscular junction (NMJ) abnormalities in Drosophila melanogaster models of ALS expressing the human FUSP525L mutant. Moreover, we demonstrate that the increase in m6A is attributable to reduced expression of the m6A eraser ALKBH5, resulting from impaired translation caused by direct FUS binding to its transcript. We further show that ALKBH5 downregulation contributes to RNA and protein alterations observed in FUS-associated ALS, driving the abnormal SG dynamics characteristic of the disease. This study supports the hypothesis that restoring correct m6A levels could serve as a potential therapeutic strategy for ALS.

molecular biology↗

bioRxiv2024

Long noncoding RNA HSCHARME promotes the maturation of stem cells-derived cardiomyocytes by controlling gene regulatory networks altered in human cardiomyopathies

A growing body of evidence suggests that tissue-specific long noncoding RNAs (lncRNA) play pivotal roles in the heart. Here, we exploited the synteny between the mouse and human genomes to identify the novel lncRNA HSCHARME (Human Syntenic CHARME) and combined single-cell transcriptomics, CAGE-seq data, RNA-FISH imaging and CRISPR-Cas9 genome editing to document its role in cardiomyogenesis. By investigating the mechanism of action of HSCHARME in hiPSC-derived cardiomyocytes, we found that the locus produces the major pCHARME isoform that associates with SC35-containing speckles and interacts with the splicing regulator PTBP1. Consistently, the functional inactivation of pCHARME influences the splicing of cardiac-specific pre-mRNAs and impacts their expression, which parallels a decline in cardiomyocyte differentiation and physiology. In line with a possible association with disease, large-scale analysis of the lncRNA expression across cardiomyopathy patients revealed increased levels of pCHARME in hypertrophic (HCM) and dilated (DCM) hearts and identified a subset of disease-associated targets whose expression can be modulated through HSCHARME dosage. By unlocking mechanistic insights into the role of pCHARME in cardiac cells, our data identify a novel non-coding regulator of cardiomyocyte function with potential implications in disease.

molecular biology↗

bioRxiv2024

An open-source MRI compatible frame for multimodal presurgical mapping in macaque and capuchin monkeys

HighlightsO_LIWe present a compact MRI-compatible stereotaxic frame for large nonhuman primates. C_LIO_LIThe design is 3D printable, inexpensive, and matches size of an adult human head. C_LIO_LIEnabled real-time, accurate, MRI-guided deep-brain viral vector injection. C_LIO_LIFacilitated multimodal alignment for deep-brain electrophysiology planning. C_LIO_LIAll computer-aided-design files are modularized and publicly available and editable. C_LI BackgroundHigh-precision neurosurgical targeting in nonhuman primates (NHPs) often requires presurgical anatomy mapping with noninvasive neuroimaging techniques (MRI, CT, PET), allowing for translation of individual anatomical coordinates to surgical stereotaxic apparatus. Given the varied tissue contrasts that these imaging techniques produce, precise alignment of imaging-based coordinates to surgical apparatus can be cumbersome. MRI-compatible stereotaxis with radiopaque fiducial markers offer a straight-forward and reliable solution, but existing commercial options do not fit in conformal head coils that maximize imaging quality. New methodWe developed a compact MRI-compatible stereotaxis suitable for a variety of NHP species (Macaca mulatta, Macaca fascicularis, and Cebus apella) that allows multimodal alignment through technique-specific fiducial markers. Comparison with existing methodsWith the express purpose of compatibility with clinically available MRI, CT, and PET systems, the frame is no larger than a human head, while allowing for imaging NHPs in the supinated position. This design requires no marker implantation, special software, or additional knowledge other than the operation of a common large animal stereotaxis. ResultsWe demonstrated the applicability of this 3D-printable apparatus across a diverse set of experiments requiring presurgical planning: 1) We demonstrate the accuracy of the fiducial system through a within-MRI cannula insertion and subcortical injection of viral vectors. 2) We also demonstrated accuracy of multimodal (MRI and CT) alignment and coordinate transfer to guide a surgical robot electrode implantation for deep-brain electrophysiology. ConclusionsThe computer-aided design files and engineering drawings are publicly available, with the modular design allowing for low cost and manageable manufacturing.

bioengineering↗

bioRxiv2023

A (Sub)field Guide to Quality Control in Hippocampal Subfield Segmentation on High-resolution T2-weighted MRI

Inquiries into properties of brain structure and function have progressed due to developments in magnetic resonance imaging (MRI). To sustain progress in investigating and quantifying neuroanatomical details in vivo, the reliability and validity of brain measurements are paramount. Quality control (QC) is a set of procedures for mitigating errors and ensuring the validity and reliability of brain measurements. Despite its importance, there is little guidance on best QC practices and reporting procedures. The study of hippocampal subfields in vivo is a critical case for QC because of their small size, inter-dependent boundary definitions, and common artifacts in the MRI data used for subfield measurements. We addressed this gap by surveying the broader scientific community studying hippocampal subfields on their views and approaches to QC. We received responses from 37 investigators spanning 10 countries, covering different career stages, and studying both healthy and pathological development and aging. In this sample, 81% of researchers considered QC to be very important or important, and 19% viewed it as fairly important. Despite this, only 46% of researchers reported on their QC processes in prior publications. In many instances, lack of reporting appeared due to ambiguous guidance on relevant details and guidance for reporting, rather than absence of QC. Here, we provide recommendations for correcting errors to maximize reliability and minimize bias. We also summarize threats to segmentation accuracy, review common QC methods, and make recommendations for best practices and reporting in publications. Implementing the recommended QC practices will collectively improve inferences to the larger population, as well as have implications for clinical practice and public health.

neuroscience↗

bioRxiv2022

The long noncoding RNA Charme supervises cardiomyocytes maturation by controlling cell differentiation programs in the developing heart

Long noncoding RNAs (lncRNAs) are emerging as critical regulators of heart physiology and disease, although the studies unveiling their modes-of-action are still limited to few examples. We recently identified pCharme, a chromatin-associated lncRNA whose functional knockout in mice results in defective myogenesis and morphological remodelling of the cardiac muscle. Here, we combined Cap-Analysis of Gene Expression (CAGE), single-cell (sc)RNA sequencing and whole-mount in situ hybridization analyses to study pCharme cardiac expression. Since the early steps of cardiomyogenesis, we found the lncRNA being specifically restricted to cardiomyocytes, where it assists the formation of specific nuclear condensates containing MATR3, as well as important RNAs for cardiac development. In line with the functional significance of these activities, pCharme ablation in mice results in a delayed maturation of cardiomyocytes, which ultimately leads to morphological alterations of the myocardium and ventricular hypo-trabeculation. Since congenital anomalies in myocardium are clinically relevant in humans and predispose patients to major complications, the identification of novel genes controlling cardiac morphology becomes crucial. Our study offers unique insights into a novel lncRNA-mediated regulatory mechanism promoting cardiomyocyte maturation and bears relevance to Charme locus for future theranostic applications.

molecular biology↗

bioRxiv2021

Limiting the loss of terrestrial ecosystems to safeguard nature for biodiversity and humanity

Humanity is on a pathway of unsustainable loss of the natural systems upon which we, and all life, rely. To date, global efforts to achieve internationally-agreed goals to reduce carbon emissions, halt biodiversity loss, and retain essential ecosystem services, have been poorly integrated. However, these different goals all rely on preserving natural ecosystems. Here, we show how to unify these goals by empirically deriving spatially-explicit, quantitative area-based targets for the retention of natural terrestrial ecosystems. We found that at least 67 million km2 of Earths natural terrestrial ecosystems (~79% of the area remaining) require retention - via a combination of strict protection but more prominently through sustainably managed land use regimes complemented by restoration actions - to contribute to biodiversity, climate, soil and freshwater objectives under four United Nations Resolutions. This equates to retaining natural ecosystems across ~50% of the total terrestrial (excluding Antarctica) surface of Earth. Our results show where retention efforts could be focussed to contribute to multiple goals simultaneously. The retention targets concept that we present explicitly recognises that such management can and should co-occur alongside and be driven by the people who live in and rely on places where natural and semi-natural ecosystems remain on Earth.

ecology↗

bioRxiv2020

Analysis of Hippocampal Subfields in Sickle Cell Disease Using Ultrahigh Field MRI

Sickle cell disease (SCD) is an inherited hemoglobinopathy that causes organ dysfunction, including cerebral vasculopathy and neurological complications. Hippocampal segmentation with newer and advanced 7 Tesla (7T) MRI protocols has revealed atrophy in specific subregions in other neurodegenerative and neuroinflammatory diseases, however, there is limited evidence of hippocampal involvement in SCD. Thus, we explored whether SCD may be also associated with abnormalities in hippocampal subregions. We conducted 7T MRI imaging in individuals with SCD, including the HbSS, HbSC and HbS/beta thalassemia genotypes (n=37), and healthy race and age-matched controls (n=40), using a customized head coil. Both T1 and T2 weighted images were used for automatic segmentation of the hippocampus subfields. Individuals with SCD had significantly smaller volume of the Dentate Gyrus and Cornu Ammonis (CA) 2 and 3 as compared to the control group. Other hippocampal subregions also showed a trend towards smaller volumes in the SCD group. These findings support previous reports of reduced volume in the temporal lobe in SCD patients. Further studies are necessary to investigate the mechanisms that lead to structural changes in the hippocampus subfields and their relationship with cognitive performance in SCD patients.

neuroscience↗

bioRxiv2020

Improved 7 Tesla Transmit Field Homogeneity with Reduced Electromagnetic Power Deposition Using Coupled Tic Tac Toe Antennas

Recently cleared by the FDA, 7 Tesla (7T) MRI is a rapidly growing technology that can provide higher resolution and enhanced contrast in human MRI images. However, the increased operational frequency (~297 MHz) hinders its full potential since it causes inhomogeneities in the images and increases the power deposition in the tissues. This work describes the optimization of an innovative radiofrequency (RF) head coil coupled design, named Tic Tac Toe, currently used in large scale human MRI scanning at 7T; to date, this device was used in more than 1,300 patient/volunteer neuro 7T MRI scans. Electromagnetic simulations were performed for each of the coils antennas using the finite-difference time-domain method. Numerical optimizations were used to combine the calculated electromagnetic fields produced by these antennas, based on the superposition principle, and successfully produced homogeneous magnetic field distributions at low levels of power deposition in the tissues. The simulations were then successfully validated in-vivo using the Tic Tac Toe RF head coil system on a 7T MRI scanner.

bioengineering↗

bioRxiv2020

Epigenetic regulation of Wnt7b expression by the cis-acting long noncoding RNA lnc-Rewind in muscle stem cells

Skeletal muscle possesses an outstanding capacity to regenerate upon injury due to the adult muscle stem cells (MuSCs) activity. This ability requires the proper balance between MuSCs expansion and differentiation which is critical for muscle homeostasis and contributes, if deregulated, to muscle diseases. Here, we functionally characterize a novel chromatin-associated lncRNA, lnc-Rewind, which is expressed in murine MuSCs and conserved in human. We find that, in mouse, lnc-Rewind acts as an epigenetic regulator of MuSCs proliferation and expansion by influencing the expression of skeletal muscle genes and several components of the WNT (Wingless-INT) signalling pathway. Among them, we identified the nearby Wnt7b gene as a direct lnc-Rewind target. We show that lnc-Rewind interacts with the G9a histone lysine methyltransferase and mediates the in cis repression of Wnt7b by H3K9me2 deposition. Overall, these findings provide novel insights into the epigenetic regulation of adult muscle stem cells fate by lncRNAs.

cell biology↗