bluuebunny/crossref_metadata_2025 huggingface dataset:
Cannabinoids shift the basal ganglia microRNA m6A methylation profile towards an anti-inflammatory phenotype in SIV-infected rhesus macaques
Authors: Okeoma Chioma M. , Premadasa Lakmini S. , Tan Chen S. , Ghiran Ionita C. , Mohan Mahesh
Published: October 2024

Background

Epitranscriptomic modifications modulate diverse biological processes like regulation of gene expression, abundance, location and function. N6-methyladenosine (m6A) methylation has been shown to regulate various diseases, including cancer and inflammation. While there is evidence that m6A modification is functionally relevant in neural development and differentiation, the role of m6A modification in HIV neuropathogenesis is unknown.

Methods

Here, we used anti-N6-methyladenosine (m6A) antibody immunoprecipitation and microarray profiling to identify m6A modifications in miRNAs in basal ganglia (BG) of Rhesus macaques (RMs) that were uninfected (VEH) and SIV-infected on combination anti-retroviral therapy (cART) and either VEH-treated (VEH/SIV/cART), or THC:CBD-treated (THC:CBD/SIV/cART). Ingenuity pathway analysis was conducted to understand the biological implications of miRNA m6A methylation in HIV neuropathogenesis. Finally, to understand the functional significance of m6A modifications in miRNAs, we overexpressed FAM-labeled wild-type or m6A-modified miR-194-5p in SCC-25 cells and determined its impact on the expression of its target, STAT1, an interferon-stimulated transcription factor known to drive persistent neuroinflammation in several neurodegenerative diseases.

Results

HIV/SIV infection promoted an overall hypomethylated miRNA m6A profile. While the overall hypomethylated m6A profile was not significantly impacted by THC:CBD, specific miRNAs predicted to target proinflammatory genes showed markedly reduced m6A methylation levels compared to the VEH-treated RMs. Additionally, specific BG tissue miRNAs bearing m6A epi-transcriptomic marks were transferred and detected in BG-derived extracellular vesicles. Mechanistically, the DRACH motif in the seed region of miR-194-5p was significantly m6A hypomethylated in THC:CBD/SIV/cART RMs. In SCC-25 cells, unlike wild-type miR-194-5p, transfected m6A-modified miR-194-5p mimics failed to downregulate STAT1 protein expression. Further, compared to VEH/SIV/cART RMs, THC:CBD administration significantly reduced m6A methylation of 44 miRNAs directly involved in regulating CNS network genes.

Conclusions

These results underscore the need for investigating the qualitative, and posttranscriptional modifications in RNA along with the more traditional, quantitative alterations in pathological conditions or in response to disease modifying treatments. Our findings indicate that m6A epitranscriptomic marks in the seed nucleotide region can impair miRNA function and that cannabinoids may preserve it by reducing m6A methylation levels. Finally, these findings provide a novel mechanistic (miRNA m6A hypomethylation) explanation underlying the anti-neuroinflammatory effects of phytocannabinoids in HIV/SIV infection.
Evidence forin vivomRNA Transport Between Mammalian Cells
Authors: Shubin Andrey , Liu Songlei , Church George , Hunter Craig
Published: October 2024

The prevalence and significance of intercellular mRNA transport remains unknown. Direct detection of mRNA transfer between cells within an organism is challenging due to technical limitations associated with transgene encoded molecular labels and cell sorting techniques. In this study we analyzed human-to-mouse xenograft single-cell RNA sequencing data to identify mouse transcripts in recovered human cells. The murine transcriptome analysis implicates macrophages as a frequent mRNA donor cell type. We then developed anin vitrosystem using mouse RAW264.7 macrophages and human HeLa and HEK293 cells and confirmed the transfer of Ftl1 mRNA from mouse to human cells. Overall, our study provides compellingin vivoevidence for prevalent intercellular mRNA transfer in human-to-mouse xenograft models.
Novel correlative microscopy approach for nano-bio interface studies of ultrafine particle-induced lung epithelial cell damage
Authors: Podlipec Rok , Pirker Luka , Krišelj Ana , Hlawacek Gregor , Gianoncelli Alessandra , Pelicon Primož
Published: October 2024

Correlative light and electron microscopy (CLEM) has attracted widespread interest in the life sciences community, particularly in light of recent developments in imaging resolution and sensitivity, and sample preservation. One of the highly relevant scientific areas that can be addressed by CLEM is nanotoxicology, the study of the environmental and, in particular, health effects of the ubiquitous particulate matter in polluted air. To assess safety and predict outcomes, particularly those induced by highly penetrating ultrafine particles (UFPs), it is invaluable to understand the mode of action at the molecular level, from the moment the particles interact with lung tissue. In our study we address these challenges by combining novel multimodal high-resolution live and fixed cell microscopy, from fluorescence lifetime imaging microscopy (FLIM), hyperspectral fluorescence imaging (fHSI), scanning electron microscopy (SEM), ultra-high resolution helium ion microscopy (HIM) to synchrotron micro X-ray fluorescence (SR µXRF). These provided a comprehensive insight into the structural and functional properties of the nano-bio interface formed on the surface of lung epithelial cells exposed to titanium dioxide nanotubes (TiO2NTs) and for the first time, we report several important initial cellular responses. Besides the characterization of extensive biomolecule binding to TiO₂, including DNA and its accumulation-related plausible implications for apoptosis and inflammation, we show acute changes due to fibrin-like fibrous network formation over aggregated nano-bio composites and accumulated iron-induced changes in the molecular environment at the nano-bio interface. The applied CLEM approach demonstrates high applicability for studying the initial cellular responses to nanoparticles, which can play a crucial role in the body’s immune response and the development of inflammation. The methodology provides a robust framework for future research into nanoparticle toxicity and its impact on human health.
RNA Polymerase II is a Polar Roadblock to a Progressing DNA Fork
Authors: Kay Taryn M. , Inman James T. , Lubkowska Lucyna , Le Tung T. , Qian Jin , Hall Porter M. , Wang Dong , Kashlev Mikhail , Wang Michelle D.
Published: October 2024

DNA replication and transcription occur simultaneously on the same DNA template, leading to inevitable conflicts between the replisome and RNA polymerase. These conflicts can stall the replication fork and threaten genome stability. Although numerous studies show that head-on conflicts are more detrimental and more prone to promoting R-loop formation than co-directional conflicts, the fundamental cause for the RNA polymerase roadblock polarity remains unclear, and the structure of these R-loops is speculative. In this work, we use a simple model system to address this complex question by examining the Pol II roadblock to a DNA fork advanced via mechanical unzipping to mimic the replisome progression. We found that the Pol II binds more stably to resist removal in the head-on configuration, even with minimal transcript size, demonstrating that the Pol II roadblock has an inherent polarity. However, an elongating Pol II with a long RNA transcript becomes an even more potent and persistent roadblock while retaining the polarity, and the formation of an RNA-DNA hybrid mediates this enhancement. Surprisingly, we discovered that when a Pol II collides with the DNA fork head-on and becomes backtracked, an RNA-DNA hybrid can form on the lagging strand in front of Pol II, creating a topological lock that traps Pol II at the fork. TFIIS facilitates RNA-DNA hybrid removal by severing the connection of Pol II with the hybrid. We further demonstrate that this RNA-DNA hybrid can prime lagging strand replication by T7 DNA polymerase while Pol II is still bound to DNA. Our findings capture basal properties of the interactions of Pol II with a DNA fork, revealing significant implications for transcription-replication conflicts.
Integration of spatial transcriptomics with immunofluorescence staining reveals spatial heterogeneity and plasticity of astrocytes in experimental glioblastomas
Authors: Ghosh Mitrajit , Pilanc-Kudlek Paulina , Jacek Karol , Baluszek Szymon , Poleszak Katarzyna , Szadkowska Paulina , Gielniewski Bartłomiej , Miklaszewska Aleksandra Ellert , Kaminska Bozena
Published: October 2024

Astrocytes comprise ∼50% of all brain cells and present distinct morphological, molecular and functional properties in different brain regions. In glioblastoma (GBM), an aggressive primary brain tumour, tumour-associated astrocytes (TAAs) become activated and exhibit different transcriptomic profiles, morphology and functions supporting disease progression. Heterogeneity and specific roles of TAAs within various regions of tumours are poorly known. Advancements of single-cell and spatial transcriptomics allow to profile tumours at unprecedented resolution revealing cell phenotypes, hidden functionalities and spatial architecture in disease-specific context.

We combined spatial transcriptomics and multiple immunofluorescent staining to visualize TAAs heterogeneity and location of various subpopulations in intracranial murine gliomas. Using distinct gene expression profiles, we identified subtypes of TAAs with distinct localization and inferred their specialized functionalities. Gene signatures associated with TAAs reflected their reprograming in the tumour microenvironment (TME), revealed their multiple roles and potential contributing factors shaping the local milieu. Using spatial correlation analysis of the spots, we inferred the interactome ofSlc1a2(encoding a glutamate transporter) with the other markers of TAAs based on segregated areas of the tumour. The designer RGD peptide blocking tumour-microglia communications, alters the spatial distribution of TAAs in experimental gliomas providing insights into potential mechanisms. Spatial transcriptomics combined with multiple staining unveils multiple functional phenotypes of TAAs and interactions within TME. It shows their distinct morphology and unveils different roles in various regions of the tumour. We demonstrate the glioma-induced heterogeneity of TAAs and their adaption to the pharmacologically-induced modification of the immunosuppressive TME.
Neuroanatomy of the Accessory Olfactory Bulb in the Fossorial Water Vole
Authors: Ruiz-Rubio Sara , Ortiz-Leal Irene , Torres Mateo V. , Elsayed Mostafa G. A. , Somoano Aitor , Sanchez-Quinteiro Pablo
Published: October 2024

The accessory olfactory bulb (AOB) plays a key role in processing chemical signals crucial for species-specific social and reproductive behaviors. While extensive research has focused on the vomeronasal system of laboratory rodents, less is known about wild species, particularly those that rely heavily on chemical communication. This study aims to characterize the morphological and neurochemical organization of the AOB in the fossorial water vole (Arvicola scherman), a subterranean rodent species from the family Cricetidae. We have employed histological techniques, including Nissl and hematoxylin staining, as well as immunohistochemical and lectin-histochemical markers, to assess the AOB structure. Our findings reveal that the AOB of the water vole exhibits a distinct laminar organization with prominent mitral cells in the mitral-plexiform layer, as well as dense labeling of periglomerular and short-axon cells in the glomerular layer. Lectin histochemistry further confirmed zonation patterns analogous to those seen in other rodent species. Immunohistochemical analysis demonstrated significant expression of PGP 9.5, suggesting its involvement in maintaining neuronal activity within the AOB. In contrast, the absence of SMI-32 labeling in the AOB, compared to its strong expression in the main olfactory bulb, highlights functional distinctions between these two olfactory subsystems. These structural and neurochemical characteristics suggest that the AOB of the fossorial water vole is adapted for enhanced processing of chemosensory signals, which may play a pivotal role in its subterranean lifestyle. Our results provide a foundation for future studies exploring the functional implications of these adaptations, including potential improvements in the integrated management of these vole populations.
Deciphering gene regulatory programs in mouse embryonic skin through single-cell multiomics analysis
Authors: Deng Qiuting , Cai Pengfei , Luo Yingjie , Zhang Zhongjin , Ma Wen , Huang Zijie , Chen Xiaoya , Hao Shijie , Ma Weiguang , Xu Jiangshan , Cheng Mengnan , Lin Xiumei , Zhou Ru , Duan Shanshan , Chen Junjie , Li Ronghai , Shi Xuyang , Liu Chang , Gao Peng , Li Jianting , Xie Jun , Liu Longqi , Yuan Yue , Liu Chuanyu
Published: October 2024

Background

Cell type-specific transcriptional heterogeneity in embryonic mouse skin is well-documented, but few studies have investigated the regulatory mechanisms.

Results

Here, we present high throughput single-cell chromatin accessibility and transcriptome sequencing (HT-scCAT-seq), a method that simultaneously profiles transcriptome and chromatin accessibility. We utilized HT-scCAT-seq to dissect the gene regulatory mechanism governing epidermal stratification, periderm terminal differentiation, and fibroblast specification.

Conclusions

By linking chromatin accessibility to gene expression, we identified candidatecis-regulatory elements (cCREs) and target genes crucial for dermal and epidermal development. We described cells with similar gene expression profiles that exhibit distinct chromatin accessibility statuses during periderm terminal differentiation. Finally, we characterized the underlying lineage-determining transcription factors (TFs), and demonstrated that ALX4 and RUNX2 were candidate TF regulators of the dermal papilla lineage development through in silico perturbation analysis.
Lipidomic Profiling Reveals Shared and Distinct Pathological Signatures in Sporadic Parkinson’s Disease and GBA Mutation Carriers: Implications for Disease Mechanisms
Authors: Muñoz Sonia Sanz , Marlet Frederik Ravnkilde , Bilgin Mesut , Dreier Jesper E , Bezard Erwan , Dehay Benjamin , Jaunmuktane Zane , Maeda Kenji , Galvagnion Céline
Published: October 2024

Parkinson’s Disease (PD) is a neurodegenerative disorder characterised by the deposition of protein inclusions, called Lewy Bodies (LBs), in neurons. LBs are heterogeneous structures whose main constituent is the protein alpha-synuclein (αS) and that are also composed of lipid molecules. Disruptions in the levels of specific lipids, including sphingolipids, fatty acids, and cholesterol, have been associated with PD, suggesting a role of lipids in the emergence and spreading of αS and PD pathology. Using a combination of shotgun lipidomics and biochemical analyses of PD amygdala homogenates, we have shown that long sporadic disease duration andGBArisk mutation are associated with a decrease in the protein and activity levels of glucocerebrosidase (GCase) activity and in cardiolipin levels and an increase in those of pathological αS, cholesterol, diacylglycerides, sphingolipids and specific glycerophospholipids (GPL). Long sporadic PD and GBA risk mutations also led to a shift from long unsaturated to short saturated GPL and from short to long sphingomyelin and ceramide. Moreover, the levels of lipid classes and species affected by long sPD andGBArisk mutations were found to correlate negatively with GCase activity and positively with pathological αS levels. We found thatGBAmutation with mild phenotype affects lipid levels in the same direction asGBArisk mutation and long sPD but to a lesser extent and thatGBAmutation carriers with severe phenotype led to changes in the opposite direction for the same lipids. Finally, the lipid analyses of LB- and small aggregates enriched fractions show that long sPD andGBArisk mutations led to the same changes in the levels and species distribution of GPL and SL than in homogenates but to a lesser extent. Together, these results suggest the need for patient stratification in clinical trials of therapeutic interventions in PD-GBA and that successful therapeutics against PD-GBA should be considered for sporadic PD.
The neuropeptide aCGRP impairs the chondrocyte response to mechanical load
Authors: Dietmar H.F. , Hecht N. , Diederichs S.
Published: October 2024

Mechanical loading can contribute to articular cartilage homeostasis due to its anabolic effects, for example the stimulation of glycosaminoglycan (GAG) synthesis. During degenerative joint diseases, such as osteoarthritis, the ability to respond adequately to mechanical stimulation may be compromised. The neuropeptide and pain mediator αCGRP has previously been associated with osteoarthritis and is typical for sensory nerve fibres, but chondrocytes also possess the receptors for αCGRP and have been shown to respond to treatment in vitro. Since it was reported that murine macrophages were more sensitive to αCGRP following stretching, we hypothesised that αCGRP may influence the response of chondrocytes to mechanical stimulation. Here, we employed our previously established model system using human articular chondrocytes to generate tissue-engineered cartilage to investigate the effect of the sensory neuropeptide αCGRP on the chondrocyte loading response. Following culture under chondrogenic conditions for 35 days, tissue-engineered cartilage was subjected to a three-hour episode of unconfined dynamic compression in the presence or absence of αCGRP. Addition of αCGRP did not exert obvious anti-chondrogenic effects, and typical mechano-response markers were not altered when mechanical loading was performed in the presence of αCGRP. GAG synthesis rates were stimulated by mechanical loading in the control group as expected, but this anabolic effect was prevented by the presence of αCGRP. Our results suggest that high levels of αCGRP may interfere with the mechano-adaptation of articular cartilage and this may have important implications for osteoarthritis pathology and therapy.
Thermoceptive predictions and prediction errors in the anterior insula
Authors: Toussaint Birte , Heinzle Jakob , Friedli Nicole , Zahnd Nicole Jessica , Federici Elena , Köchli Laura , Harrison Olivia Kate , Iglesias Sandra , Stephan Klaas Enno
Published: October 2024

Contemporary theories of interoception propose that the brain constructs a model of the body for predicting the states and allostatic needs of all organs, including the skin, and updates this model using prediction error signals. However, empirical tests of this proposal are scarce in humans. This computational neuroimaging study investigated the presence and location of thermoceptive predictions and prediction errors in the brain using probabilistic manipulations of skin temperature in a novel interoceptive learning paradigm. Using functional MRI in healthy volunteers, we found that a Bayesian model provided a better account of participants’ skin temperature predictions than a non-Bayesian model. Further, activity in a network including the anterior insula was associated with trial-wise predictions and precision-weighted prediction errors. Our findings provide further evidence that the anterior insula plays a key role in implementing the brain’s model of the body, and raise important questions about the structure of this model.
Characterizing dynamic functional connectivity in congenitally blind people using Hidden Markov Models
Authors: Pedersini Caterina Annalaura , Rokers Bas , Collignon Olivier
Published: October 2024

How does sensory experience influence the brain connectome? Early acquired blindness is known to trigger a large-scale alteration in brain connectivity. Previous studies have traditionally assessed stationary functional connectivity across all timepoints in scanning sessions. In this study we compared the dynamic nature of functional connectivity in sighted (50) and early/congenitally blind (33) individuals through the data-driven approach of Hidden Markov Models (HMM). Blind individuals showed increased fractional occupancy in two key brain states: one characterized by bilateral somatomotor activation and another by occipito-frontal activation, mainly in the left hemisphere. Conversely, sighted individuals more frequently occupied a state associated with Default Mode and Ventral Attentional networks. Network analysis revealed decreased connectivity in blind individuals within states frequently occupied by sighted individuals, particularly between visual and somatomotor regions. In contrast, states more often visited by blind individuals exhibited increased connectivity within the visual network, connecting multiple occipital regions with the temporal fusiform area, as well as between visual and prefrontal regions. These results suggest that the frequency of visiting specific brain states may contribute to the differences observed between blind and sighted individuals in stationary resting state functional connectivity. Together, these findings show the effectiveness of Hidden Markov Models in capturing the temporal dynamics of functional connectivity, revealing its variability over time. We found that early visual deprivation disrupts these dynamics, with blindness leading to prolonged activation of unimodal regions and increased connectivity within the visual network and between occipital and higher-order brain regions. Additionally, alterations in the chronnectome, defined as the functional connectome changing over time - specifically in the time spent visiting a state - may provide new insights into stationary functional connectivity patterns in blind individuals, establishing a foundation for understanding network connectivity dynamics following sensory deprivation.
Simultaneous quantification of oxygen and glucose consumption during visual stimulation in the human cortex
Authors: Bose Antonia , Epp Samira Maria , Belenya Roman , Kurcyus Katarzyna , Dominguez Eric Ceballos , Ranft Andreas , Villa Eliana Salas , Bursche Moritz , Preibisch Christine , Castrillón Gabriel , Riedl Valentin
Published: October 2024

The brain relies on oxidized glucose as its primary fuel. Despite robust coupling of cerebral oxygen and glucose consumption during rest, the oxygen to glucose index (OGI) has been suggested to drop significantly during neuronal activation. However, empirical evidence regarding the extent of this uncoupling is scarce, mainly due to the inability of previous studies to measure CMRO2and CMRglcconcurrently during tasks. Therefore, in the present study, we integrated multiparametric quantitative BOLD (mqBOLD) with functional PET (fPET) to simultaneously quantify cerebral oxygen and glucose metabolism during visual stimulation and rest within a single session. Results show increases in both CMRO2and CMRglcin visual areas, concomitant with focal blood flow increases. Moreover, OGI values during rest were close to the theoretical value of 6, which is in line with previous literature. In response to visual stimulation, the OGI decreased by 6.6-21.6%, depending on the mask applied. For the first time, the present study demonstrates the feasibility of combining mqBOLD and fPET to study CMRO2and CMRglcsimultaneously. This setup has the potential to be applied to various experimental settings, providing valuable information about the extent of oxidative glucose metabolism in the human brain under different conditions in health and disease.
Dissecting the nutritional regulations of a whole amino acid transporter family from a complex genome species: A holistic approach turning weaknesses into strengths
Authors: Garrec Soizig Le , Pinel Karine , Heraud Cécile , Ganot Akintha , Veron Vincent , Morin Guillaume , Iorfida Ophélie , Cardona Emilie , Marandel Lucie , Devin Anne , Labonne Jacques , Averous Julien , Bruhat Alain , Seiliez Iban , Beaumatin Florian
Published: October 2024

Amino acid transporters (AATs) are described as pivotal in maintaining circulating and cellular concentrations of AA via regulation of their expression in response to the cellular environment. Rainbow trout (RT), a complex genome species, is poorly described for AATs roles in controlling its predominant AA-based metabolism, despite representing a major challenge in the aquaculture nutrition field. Therefore, we identified the whole repertoire of AAT found in RT genome (>200), its expression in tissues and its nutritional regulationsin vitro. Results garnered revealed the existence of different clusters of AATs, notably due to promoters bearing ATF4-related AA response elements. Moreover, the modeling of each AAT-specific cluster activities disclosed mTOR-related signaling functions of Ile and Phe, yet unknown in RT. Thus, this novel approach herein described should help to better grasp AA homeostasis in most organisms and topics such as fish nutrition and evolution.
Identification of a specialized lipid barrier forDrosophilametamorphosis
Authors: Lampe Lena , Newell Clare L. , Wang Bing-Jun , Makki Rami , Alexandre Cyrille , Gilmore Ian S. , Zhao Li , Gould Alex P.
Published: October 2024

In many terrestrial insects, the onset of metamorphosis marks a transition from humid to dry environments. Yet how metamorphosing insect pupae protect themselves against the threat of dehydration remains unclear. Here, we identify the chemical composition and biosynthetic origins of a lipid desiccation barrier specific to the pupal and sexually-immature adult stages ofDrosophila melanogaster. This barrier comprises unisex hyper-long hydrocarbons, 29-37 carbons in length, which are synthesized by larval oenocytes and stored in the larval fat body before being deployed on the pupal and young adult cuticles. We show that the fatty acid elongaseEloHLis required for the biosynthesis of hyper-long hydrocarbons that are essential for the barrier to water loss during metamorphosis. Across theDrosophilagenus, many species express unisex profiles of hyper-long hydrocarbons and, as young adults, transition to sex-specific shorter hydrocarbons with known pheromonal functions. The desert speciesD. mojavensis, however, retains hyper-long hydrocarbons during adulthood likely as an adaptation to an arid environment. Our study reveals how the cuticular lipid barrier is tuned to meet changing environmental pressures during insect development and evolution.
Amyloid beta precursor protein contributes to brain aging and learning decline in short-lived turquoise killifish (Nothobranchius furzeri)
Authors: de Bakker Dennis E.M. , Mihaljević Mihaela , Gharat Kunal , Richter Yasmin , Bagnoli Sara , van Bebber Frauke , Adam Lisa , Shamim-Schulze Farzana , Ohlenschläger Oliver , Bens Martin , Cirri Emilio , Antebi Adam , Matić Ivan , Schneider Anja , Schmid Bettina , Cellerino Alessandro , Kirstein Janine , Valenzano Dario Riccardo
Published: October 2024

Brain aging is a pivotal risk factor for many neurodegenerative diseases, yet its molecular and cellular mechanisms remain elusive. The amyloid-beta precursor protein (APP) is among the most studied proteins linked to brain pathology; however, its role in non-pathological brain aging remains poorly characterized. Here, we investigate the natural impact of APP on normal brain aging using the short-lived turquoise killifish (Nothobranchius furzeri), which exhibits rapid and spontaneous age-related decline. We found that pyroglutamated amyloid beta —a neurotoxic Aβ variant—accumulates intra-neuronally in an age-dependent manner, co-localizing with a marker of cell death. We found that intraneuronal pyroglutamated amyloid beta is also present in old human brains, suggesting deep evolutionary conservation. To determine Aβ’s role in spontaneous brain aging, we generated an “amyloid precursor protein a” (appa) knock-out killifish line using CRISPR/Cas9. The knock-out ofapparescued age-dependent increase in cell death and inflammation, mitigated proteome-wide brain aging and improved the age-related decline in neuronal activity and learning capacity. Our findings show a key role for Aβ precursor protein in non-pathological brain aging, making it a suitable target for anti-aging interventions.
Syngeneic adipose-derived stromal cells modulate the immune response but have limited persistence within decellularized adipose tissue implants in C57BL/6 mice
Authors: Walker John T. , Cooper Tyler T. , Dunmore-Buyze Joy , Serack Fiona E. , Brooks Courtney , Grant Aaron , Drangova Maria , Lajoie Gilles , Dekaban Gregory A. , Flynn Lauren E.
Published: October 2024

The delivery of adipose-derived stromal cells (ASCs) on cell-instructive decellularized adipose tissue (DAT) scaffolds is a promising strategy for stimulating host-derived soft tissue regeneration. However, a better understanding of the mechanisms through which ASCs modulate regenerationin vivois needed to harness these cells more effectively. In this study, DAT scaffolds, both with and without seeded syngeneic DsRED+mouse ASCs, were implanted into immunocompetent C57BL/6 mice. Downstream analyses focused on assessing donor ASC persistence and phenotype, as well as the effects of ASC seeding on host macrophage polarization and the perfused host vascular network. Notably, most donor ASCs were cleared from the scaffolds by 2 weeks. Mass spectrometry-based proteomics indicated that the transplanted ASCs maintained their pre-implantation phenotype up to 1 weekin vivo, suggesting that the cells were not undergoing programmed cell death. A higher fraction of the infiltrating host macrophages expressed CD68 and Arginase-1 in the ASC-seeded implants up to 1-week post-implantation. Interestingly, a small population of phagocytic macrophages, identified by uptake of DsRED protein, was present in the DAT implants in the first 2 weeks and showed enhanced expression of CD68, Arginase-1, and CD163, along with reduced expression of iNOS. MicroCT angiography revealed a similar perfused vessel network in the seeded and unseeded groups at 4- and 8-weeks post-implantation. Overall, seeding with syngeneic ASCs modulated the host macrophage response to the DAT bioscaffolds at early timepoints, but did not impact long-term regenerative outcomes, potentially due to the rapid clearance of the donor cell population in this model.
Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis
Authors: Hawley Joshua , Lea Robert , Biga Veronica , Papalopulu Nancy , Manning Cerys
Published: October 2024

In the developing mouse ventral spinal cord, HES5, a transcription factor downstream of Notch signalling, is expressed as evenly spaced clusters of high HES5-expressing neural progenitor cells along the dorsoventral axis. While Notch signalling requires direct membrane contact for its activation, we have previously shown mathematically that contact needs to extend beyond neighbouring cells for the HES5 pattern to emerge. However, the presence of cellular structures that could enable such long-distance signalling was unclear. Here, we report that cellular protrusions are present all along the apicobasal axis of individual neural progenitor cells. Through live imaging, we show that these protrusions dynamically extend and retract reaching lengths of up to ∼20μm, enough to extend membrane contact beyond adjacent cells. The Notch ligand DLL1 was found to colocalise with protrusions, further supporting the idea that Notch signalling can be transduced at a distance. The effect of protrusions on the HES5 pattern was tested by reducing the density of protrusions using the CDC42 inhibitor ML141, leading to a tendency to decrease the distance between high HES5 cell clusters. However, this tendency was not significant and leaves an open question about their role in the fine-grained organisation of neurogenesis.
Topic Modeling analysis of the Allen Human Brain Atlas
Authors: Pizzini Letizia , Valle Filippo , Osella Matteo , Caselle Michele
Published: October 2024

The human brain is a complex interconnected structure controlling all elementary and high-level cognitive tasks. It is composed of many regions that exhibit specific distributions of cell types and distinct patterns of functional connections. This complexity is rooted in differential transcription. The constituent cell types of different brain regions express distinctive combinations of genes as they develop and mature, ultimately shaping their functional state in adulthood. How precisely the genetic information of anatomical structures is connected to their underlying biological functions remains an open question in modern neuroscience.

A major challenge is the identification of “universal patterns”, which do not depend on the particular individual, but are instead basic structural properties shared by all brains. Despite the vast amount of gene expression data available at both the bulk and single-cell levels, this task remains challenging, mainly due to the lack of suitable data mining tools.

In this paper, we propose an approach to address this issue based on a hierarchical version of Stochastic Block Modeling. Thanks to its specific choice of priors, the method is particularly effective in identifying these universal features. We use as a laboratory to test our algorithm a dataset obtained from six independent human brains from the Allen Human Brain Atlas. We show that the proposed method is indeed able to identify universal patterns much better than more traditional algorithms such as Latent Dirichlet Allocation or Weighted Correlation Network Analysis. The probabilistic association between genes and samples that we find well represents the known anatomical and functional brain organization. Moreover, leveraging the peculiar “fuzzy” structure of the gene sets obtained with our method, we identify examples of transcriptional and post-transcriptional pathways associated with specific brain regions, highlighting the potential of our approach.
Snapshot of 5-HT2Areceptor activation in the mouse brain via IP1detection
Authors: de la Fuente Revenga Mario , González-Maeso Javier
Published: October 2024

The distinct subjective effects that define psychedelics such as LSD, psilocybin or DOI as drug class are causally linked to activation of the serotonin 2A receptor (5-HT2AR). However, some aspects of 5-HT2AR pharmacology remain elusive, such as what molecular drivers differentiate psychedelic from non-psychedelic 5-HT2AR agonists. We developed an ex vivo platform to obtain snapshots of drug-mediated 5-HT2AR engagement of the canonical Gq/11pathway in native tissue. This non-radioactive methodology captures the pharmacokinetic and pharmacodynamic events leading up to changes in inositol monophosphate (IP1) in the mouse brain. The specificity of this method was assessed by comparing IP1levels in homogenates from the frontal cortex in DOI-treated wild-type and 5-HT2AR-KO animals compared to other brain regions, namely striatum and cerebellum. Furthermore, we encountered that head-twitch response (HTR) counts and IP1in the frontal cortex were correlated. We observed that IP1levels in frontal cortex homogenates from mice treated with LSD and lisuride vary in magnitude, consistent with LSD’s 5-HT2AR agonism and psychedelic nature, and lisuride’s lack thereof. MDMA evoked an increase of IP1signal in the frontal cortex that were not matched by the serotonin precursor 5-HTP or the serotonin reuptake inhibitor fluoxetine. We attribute differences in the readout primarily to the indirect stimulation of 5-HT2AR by MDMA via serotonin release from its presynaptic terminals. This methodology enables capturing a snapshot of IP1turnover in the mouse brain that can provide mechanistic insights in the study of psychedelics and other serotonergic agents pharmacodynamics.
Persistent Na+current couples spreading depolarization to seizures inScn8again of function mice
Authors: Aiba Isamu , Ning Yao , Noebels Jeffrey L.
Published: October 2024

Spreading depolarization (SD) is a slowly propagating wave of massive cellular depolarization that transiently impairs the function of affected brain regions. While SD typically arises as an isolated hemispheric event, we previously reported that reducing M-type potassium current (IKM) by ablation ofKcnq2in forebrain excitatory neurons results in tightly coupled spontaneous bilateral seizure-SD complexes in the awake mouse cortex. Here we find that enhanced persistent Na+current due to gain-of-function (GOF) mutations inScn8a(N1768D/+, hereafter D/+) produces a similar compound cortical excitability phenotype. Chronic DC-band EEG recording detected spontaneous bilateral seizure-SD complexes accompanied by seizures with a profound tonic motor component, which occur predominantly during the light phase and were detected at ages between P33-100. Laser speckle contrast imaging of cerebral blood flow dynamics resolved SD as a bilateral wave of hypoperfusion and subsequent hour-lasting hypoperfusion inScn8aD/+cortex in awake head-restrained mice evoked by a PTZ injection. Subcortical recordings in freely moving mice revealed that approximately half of the spontaneous cortical seizure-SD complexes arose with a concurrent SD-like depolarization in the thalamus and delayed depolarization in the striatum. In contrast, SD-like DC potential shifts were rarely detected in the hippocampus or upper pons. Consistent with the high spontaneous incidencein vivo, cortical slices fromScn8aD/+mice showed a raised SD susceptibility, and pharmacological inhibition of persistent Na+current (INaP), which is enhanced inScn8aD/+neurons, inhibited SD generation in cortical slicesex vivoas well as in head-fixed micein vivo, indicating that INaPcontributes to SD susceptibility.Ex vivoCa2+imaging studies using acute brain slices expressing genetic Ca2+sensor (Thy1-GCAMP6s) demonstrated that pharmacological activation of IKMsuppressed Ca2+spikes and SD, whereas an IKMinhibitor strongly increased the frequency of hippocampal Ca2+spikes inScn8aD/+, but not WT slices, suggesting that IKMrestrains theScn8aGOF hyperexcitability. Together, our study identifies a cortical SD phenotype inScn8aGOF mice shared with theKcnq2-cKO model of developmental epileptic encephalopathy, and reveals that an imbalance of non-inactivating inward and outward tonic membrane currents bidirectionally modulates spatiotemporal SD susceptibility.
Quiescent cell re-entry is limited by macroautophagy-induced lysosomal damage
Authors: Murley Andrew , Popovici Ann Catherine , Hu Xiwen Sophie , Lund Anina , Wickham Kevin , Durieux Jenni , Joe Larry , Koronyo Etai , Dillin Andrew
Published: October 2024

To maintain tissue homeostasis, many cells are kept in a quiescent state until prompted to divide. The re-activation of quiescent cells is perturbed with aging and may underlie declining tissue homeostasis and resiliency. The unfolded protein response regulators IRE-1 and XBP-1 are required for the re-activation of quiescent cells in developmentally L1 arrestedC. elegans.Utilizing a forward genetic screen inC. elegans, we discovered that macroautophagy targets protein aggregates to lysosomes in quiescent cells, leading to lysosome damage that prevents cell cycle re-entry in the absence of IRE-1/XBP-1. Genetic inhibition of macroautophagy and stimulation of lysosomes via the overexpression of HLH-30 (TFEB/TFE3) synergistically reduces lysosome damage. Protein aggregates are also targeted to lysosomes by macroautophagy in quiescent cultured mammalian cells, causing lysosome damage that is associated with reduced re-activation. Thus, lysosome damage is a hallmark of quiescent cells and limiting lysosome damage by restraining macroautophagy can stimulate their re-activation.
Changes in cortical grasp-related activity before and after object contact
Authors: Yan Yuke , Sobinov Anton R. , Goodman James M. , Okorokova Elizaveta V. , Miller Lee E. , Bensmaia Sliman J.
Published: October 2024

Abstract

Grasping, a seemingly simple manual behavior, requires the coordinated control of dozens of joints, guided by sensory signals from muscles, tendons, and skin. As the motor cortex controls finger movement and exerted forces, the somatosensory cortex must process the barrage of proprioceptive and tactile signals that convey details about the object’s shape, its local features (e.g., edges and curvature), and forces applied to it. In the present study, we aimed to understand the transformation in these sensorimotor signals at the time of contact with an object. We analyzed object-specific signals in the primary motor cortex (M1) and Brodmann’s areas 3a, 1, and 2 of the somatosensory cortex of macaque monkeys. We found object information distributed throughout sensorimotor cortex, some of which was independent of contact, while most was dramatically altered by it. While all areas conveyed object information after contact, those carrying postural representations (M1, area 3a) were also informative before contact, during the hand pre-shaping epoch. Although their mappings retained some similarity between epochs, decoders built on the pre-contact epoch did not perform well on the post-contact epoch, suggesting intermixing between postural and force-related signals. After contact, individual neurons in M1 retained some information about the object, but the populational encoding of object identity weakened, reflecting perhaps, the delegation of control to subcortical structures. Unexpectedly, although it was active, area 2 was uninformative about the object before contact, despite its proprioceptive inputs. However, after contact, area 2 emerged as the most informative region of any epoch, likely reflecting its convergent proprioceptive and cutaneous input, and supporting its proposed role in haptic object perception. These results underscore the diverse activity within the sensorimotor cortex during grasping, highlighting the intricate neural processes involved in this fundamental behavior.
DNA methylation either antagonizes or promotes Polycomb recruitment at transposable elements
Authors: Hure Valentin , Piron-Prunier Florence , Déléris Angélique
Published: October 2024

Transposable elements (TEs) are primarily silenced by DNA methylation and the associated histone modification H3K9me2 in many multicellular eukaryotes, including plants. However, in the absence of DNA methylation due to mutations in the DNA methylation machinery or in certain developmental contexts, the same TEs can gain Polycomb-associated H3K27me3, another epigenetic silencing mark that is usually linked with the facultative silencing of genes. In this study, we initially aimed to investigate whether DNA methylation and H3K27me3 could compete during the establishment of silencing at TEs in the model plant Arabidopsis. Strikingly, we show that the deposition of the H3K27me3 mark at newly inserted transgenic TE sequences is impaired in plants in which thede novoDNA methyltransferase DRM2 is mutated, contrary to the competition model hypothesized. Further profiling of H3K27me3 indrm2mutants and in the DNA demethylase mutantrddconfirmed this new role of DNA methylation in promoting H3K27me3 deposition at some loci, in addition to the previously described antagonistic role at others; these findings further reveal a new function of DNA demethylation in modulating H3K27me3 levels in vegetative tissues. Finally, by implementing targeted DNA methylation experiments, we demonstrate that DNA methylation has a direct impact on PcG recruitment and can either promote H3K27me3 deposition or antagonize it in a locus-specific manner. Together, our results uncover a novel crosstalk between DNA methylation and Polycomb and reveal that these two pathways, thought to be specialized and antagonistic, can be interdependent and cooperate more than anticipated to maintain genome and epigenome integrity in eukaryotes.
Characteristics of spatial summation in the magnocellular, parvocellular, and koniocellular pathways
Authors: Wu Christopher S. , Coates Daniel R.
Published: October 2024

In this study, we characterize the spatial summation properties of targeted magnocellular, parvocellular, and koniocellular pathways within the central 20° of visual field using chromatic transformations in DKL color space. For the magnocellular and koniocellular conditions, critical areas of complete spatial summation were found for all eccentricities. For the parvocellular conditions, complete spatial summation was absent within the stimulus size ranges tested. We also describe an anatomically and physiologically motivated model of receptive field pooling using probability summation. Model simulations suggest that the critical area of summation can be explained by the dendritic field size of underlying retinal ganglion cells, corroborating our psychophysical data.
LC-MS system for automatically collecting time-resolved metabolomics data of cultured cells
Authors: Chan Carly C.Y. , Groves Ryan A. , Lewis Ian A.
Published: October 2024

Temporal metabolic dynamics are a critical, but difficult to study aspect of metabolism. To address this, we developed a liquid chromatography-mass spectrometry (LC-MS) system, temporal uptake and nutritional analysis (TUNA), to automatically collect time-resolved metabolomics data of cultured cells. TUNA enables sub-minute sequential sampling, has broad metabolite coverage, supports robust metabolite identification, can monitor over 72 conditions in parallel, and can be implemented in most LC-MS laboratories. We used TUNA to monitor temporal dynamics of uropathogens (Escherichia coliandProteus mirabilis) and identify novel metabolic phenotypes that cannot be captured from a single time point.
Connected Chromatin Amplifies Acetylation-modulated Nucleosome Interactions
Authors: Li Rina , Lin Xingcheng
Published: October 2024

Histone acetylation is a key regulatory post-translational modification closely as-sociated with gene transcription. In particular, H4K16 acetylation (H4K16ac) is a crucial gene activation marker that induces an open chromatin configuration. While previous studies have explored the effects of H4K16ac on nucleosome interactions, how this local modification affects higher-order chromatin organization remains un-clear. To bridge the chemical modifications of these histone tail lysine residues to global chromatin structure, we utilized a residue-resolution coarse-grained chromatin model and enhanced sampling techniques to simulate charge-neutralization effects of histone acetylation on nucleosome stability, inter-nucleosome interactions, and higher-order chromatin structure. Our simulations reveal that H4K16ac stabilizes a single nucleosome due to the reduced entropic contribution of histone tails during DNA unwrapping. In addition, acetylation modestly weakens inter-nucleosome interactions by diminishing contacts between histone tails, DNA, and nucleosome acidic patches. These weakened interactions are amplified when nucleosomes are connected by linker DNA, whose fluctuation causes significant chromatin destacking and decompaction, exposing nucleosomes for transcriptional activity. Our findings suggest that the geometric constraint imposed by chromatin DNA plays a critical role in driving chromatin structural reorganization upon post-translational modifications.
Regulation of the SOS response and homologous recombination by an integrative and conjugative element
Authors: Heredia Alam García , Grossman Alan D.
Published: October 2024

Integrative and conjugative elements (ICEs) are mobile genetic elements that transfer between bacteria and influence host physiology and promote evolution. ICEBs1ofBacillus subtilismodulates the host DNA damage response by reducing RecA filament formation. We found that the two ICEBs1-encoded proteins, RamT and RamA that modulate the SOS response in donors also function in recipient cells to inhibit both the SOS response and homologous recombination following transfer of the element. Expression of RamT and RamA caused a decrease in binding of the host single strand binding protein SsbA to ssDNA. We found that RamA interacted with PcrA, the host DNA translocase that functions to remove RecA from DNA, likely functioning to modulate the SOS response and recombination by stimulating PcrA activity. These findings reveal how ICEBs1can modulate key host processes, including the SOS response and homologous recombination, highlighting the complex interplay between mobile genetic elements and their bacterial hosts in adaptation and evolution.