Biology subjects

Rossi, A.

Publications and source records attributed to Rossi, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Mitochondrial genome editing reveals the impact of complex I heteroplasmic variants on human neurogenesis

Mitochondrial DNA (mtDNA) variants, whether maternally inherited or acquired somatically during aging, can be particularly detrimental to cells that rely on mitochondrial metabolism such as neurons. The functional consequences of mtDNA variants are thought to depend on their heteroplasmy, defined as the proportion of mtDNA molecules carrying that variant. However, investigating the specific effects of heteroplasmic mtDNA variants on human neuronal function has been challenging. Studies using patient-derived cells cannot conclusively attribute observed phenotypes to a single mtDNA variant, while conventional genome editing approaches are not applicable to mtDNA. Here, we use DddA-derived cytosine base editor (DdCBE) to introduce pathogenic heteroplasmic mtDNA variants into Neurogenin 2 (NGN2)-inducible human induced pluripotent stem cells (iPSCs). We focus on two variants in the genes MT-ND1 and MT-ND5 belonging to complex I (CI), the largest component of the mitochondrial respiratory chain and a frequent contributor to human disease, including the incurable neurodevelopmental disorder Leigh syndrome. mtDNA-edited iPSC lines exhibit low off-target editing and maintain similar heteroplasmy following subclonal isolation and differentiation into NGN2-induced neurons (iNs). Although the CI variants do not substantially affect mitochondrial energy metabolism in undifferentiated iPSCs, they reduce mitochondrial membrane potential in iNs where they drive a transcriptional reprogramming indicative of impaired neuronal commitment. These findings suggest that heteroplasmic CI variants are sufficient to disrupt neural fate specification even at relatively low heteroplasmy. More broadly, mtDNA editing in human iPSCs provides a powerful platform to investigate how mitochondrial fitness and heteroplasmy influence development and disease.

bioengineering↗

bioRxiv2026

StabCell: Stability selection for clustering and marker detection in single-cell RNA sequencing

MotivationConventional pipelines for differential expression analysis in single-cell RNA sequencing (scRNA-seq) data first cluster individual cells and then test for differentially expressed genes between the resulting clusters. Using the same data for clustering and testing, however, poses a selective inference problem and can result in overconfidence in differences that may not reflect true biological variation. ResultsWe introduce StabCell, a stability selection framework which integrates clustering and detection of differentially expressed marker genes. By repeatedly performing clustering and differential expression analysis on complementary random subsamples, StabCell assesses clustering and marker stability, yielding a stable clustering with sets of stable marker genes. In simulations, we demonstrate that StabCell provides approximate empirical per-family error rate (PFER) control, selecting fewer false positive marker genes compared with conventional approaches, especially in cases with low signal-to-noise ratio and low sequencing depth. Applying the method to a cell differentiation dataset from induced pluripotent stem cells (IPSCs) to cardiomyocytes reveals that meaningful marker genes are consistently among the top-ranked genes. These results indicate that StabCell can improve the interpretability and robustness of scRNA-seq analyses. Availability and implementationAn implementation of StabCell in the statistical programming language R is available at https://github.com/LuckyLueck/StabCell. Code to reproduce the results is available at https://github.com/LuckyLueck/StabCell_paper.

bioinformatics↗

bioRxiv2026

Beyond Integration: Neural Dimensionality and the Landau-Ginzburg Physics of Awareness

Two-dimensional accounts of consciousness that distinguish global integration from functional diversity are empirically supported [1,2] but lack a formal phase-structure: they do not specify the nature of the transition between the two regimes, the order parameter that governs it, or the quantitative predictions that follow. We provide this structure. We propose that the dimensionality of the neural correlation structure, operationalised as the Participation Ratio of the covariance eigenspectrum, constitutes a second, independent order parameter D that governs a phase transition distinct from global integration {Phi}. Formalised through a Landau-Ginzburg free energy functional F[{Phi}, D], this transition defines a Redundant Integrated State {Delta} (high {Phi}, low D) in which globally integrated mental function is present but phenomenal experience is absent, a thermodynamic phase, not a point on a continuum. The framework generates three falsifiable predictions absent from prior work: (i) a power-law scaling D* [~] |{Phi} - {Phi}_c|^{nu} with measurable critical exponent{nu} ; (ii) a diverging susceptibility {chi}_D = {partial}D/{partial}{Phi} at the consciousness threshold, quantifiable from perturbational EEG; (iii) an explicit dissociation between MCS and VS patients in the ({Phi}, D) space, with MCS predicted to occupy state {Delta}. These predictions are directly testable with existing methodology and are not generated by any current theory of consciousness.

neuroscience↗

bioRxiv2026

Defining early human developmental identity: A curated, cross-platform marker framework

Early human development involves dynamic transitions in cell identity, including transient transcriptional modulation and stable lineage commitment. Distinguishing these types of gene expression changes is challenging and can be further exacerbated by genetic and experimental heterogeneity in the context of human pluripotent stem cell (hPSC) research. To address this challenge and help uncover transcriptional changes indicative of true developmental state, we establish a curated, cross-platform marker framework for robust identification of pluripotency and early germ-layer identity. Starting from an unbiased RNA-seq discovery set, we systematically validate candidate markers across qPCR, bulk and single-cell RNA sequencing, and quantitative proteomics platforms, yielding a refined panel of 67 markers (20 for the undifferentiated state, 17 for endoderm, 15 for ectoderm, and 15 for mesoderm). We show that this framework reliably identifies early developmental states across heterogeneous datasets, generalizes to in vivo human embryo cell types, and preserves lineage identity despite substantial transcriptional variability. Furthermore, we demonstrate concordant protein-level expression for a subset of markers, supported by deep proteomic profiling of the reference line KOLF2.1J. To enable broad application, we introduce DeepDiff, a web-based resource integrating the validated markers, allowing automated fate classification in a user-friendly interface. Together, this work provides a standardized framework for defining early human developmental identity and disentangling lineage commitment from context-dependent modulation.

developmental biology↗

bioRxiv2026

Molecular basis of tRNA modification by the human m5C methyltransferase NSUN2

RNA 5-methylcytidine (m5C) is a prevalent modification that drives RNA stability and function. In humans, m5C is deposited on distinct RNA substrates by DNMT2/TRDMT1 and the NSUN family, to regulate diverse cellular processes, but how m5C writers recognise their substrates remains unclear. NSUN2 is a major m5C methyltransferase with broad roles in cell physiology and strong links to cancer and neurodevelopmental disorders 1. Here, we reconstitute an active human NSUN2-tRNA complex and capture its post-catalytic, tRNA-bound structure at 3.1 [A] resolution. Using an integrated approach combining biochemistry, cryo-electron microscopy, crosslinking mass spectrometry and molecular dynamics simulations, we show that NSUN2 remodels the tRNA to access the variable-loop target cytidine. Recognition is driven by RNA architecture, with NSUN2 exploiting the L-shaped tRNA scaffold to position the target base in the catalytic centre. We further show that Gly679 at the NSUN2-tRNA interface is important for the stability of the complex, providing a mechanistic basis for how the disease-associated Gly679Arg substitution can impair tRNA binding. Together, these findings establish an RNA-structure-guided mechanism for NSUN2 substrate recognition and methylation and provide general principles for m5C deposition on cellular RNAs and their fundamental role in disease.

molecular biology↗

bioRxiv2026

MIC13-linked cristae disruption causes metabolic failure and early fibrotic remodelling in mitochondrial liver disease

Mitochondrial diseases are highly complex and heterogeneous, and nearly 20% of cases involve severe liver pathology. Here, we report an affected individual carrying a pathogenic MIC13 variant (c.260-2A>G) associated with early-onset mitochondrial hepato-encephalopathy. Such mitochondrial hepatopathies are rare, multisystemic disorders with major liver involvement, difficult to diagnose, lack effective treatment, and are poorly understood, in part due to the absence of faithful disease-relevant cellular models. To investigate hepatocyte-specific consequences of the MIC13 variant, we generated iPSCs carrying this disease-causing variant and differentiated them into induced hepatocytes (iHeps). MIC13, a key component of the MICOS complex required for cristae formation, was disrupted in these cells, and the resultant iHeps exhibited the same cristae defects observed in clinical samples. Integrated multi-omics and biochemical analyses revealed extensive metabolic rewiring, including disrupted amino acid turnover and accumulation of tricarboxylic acid (TCA) and urea cycle intermediates. Additionally, profound alterations in methionine cycle and transsulfuration pathways, along with enhanced bile acid synthesis, collectively affect methylation potential, redox homeostasis, and detoxification. Lipid metabolism was also impaired, with incomplete {beta}-oxidation, increased ketogenesis, and diminished lipid storage. At the cellular level, extensive extracellular matrix (ECM) remodelling, increased intracellular collagen accumulation and enhanced cell migration indicated an early fibrotic phenotype. Overall, this clinically relevant model uncovers mechanistically how cristae defects drive metabolic imbalance and hepatocyte dysfunction, ultimately leading to early fibrotic changes in mitochondrial liver disease. These findings provide a strong mechanistic foundation for understanding mitochondrial liver disease and developing targeted therapeutic strategies.

biochemistry↗

bioRxiv2025

A simple circuit to sustain intact tumor microenvironments for complex drug interrogations

Deep learning and large language models can integrate complex datasets to uncover biological insights that are often undetectable through conventional analyses. With application to translational cancer research, these computational tools have positioned 3D patient-derived tumor avatars front and center as crucial data input sources. However, a major challenge remains: the lack of standardization in media composition in 3D patient-derived tumor models unpredictably affects cell behavior and limit the utility beyond predicting treatment responses. To address this unmet need, we developed a simple, reproducible perfusion circuit system to approximate in vivo physiology using autologous patient plasma. With peritoneal metastases and core needle biopsies across multiple tumor histologies, we demonstrate preservation of the tumor microenvironment for up to 48 hours using multi-modal interrogation techniques. With proof-of-concept experiments, we display the systems ability to unveil complex drug-dependent biology within this time window. Standardizable, physiologically relevant platforms for 3D patient-derived tumor avatars will yield unprecedented insights through the integration of data from broad groups of patients and the use of an expanding armamentarium of artificial intelligence capabilities.

cancer biology↗

bioRxiv2025

Cryo-EM structure of the naked mole-rat ribosome reveals a stabilized split 28S rRNA

The naked mole-rat (Heterocephalus glaber) is a long-lived mammal with remarkable resistance to cancer and hypoxia, suggesting the evolution of robust proteostasis networks. The ribosome, the central engine of protein synthesis, is key for cellular stress responses and has an unusual feature of the 28S rRNA split; however, the details of its organization remain unknown. Here, we present high-resolution cryo-EM structures of the naked mole-rat 80S ribosome in two states of the elongation cycle. The structures reveal a conserved overall architecture and rRNA modification landscape compared to other mammals, and provide an atomic-level view of the unique break in the 28S rRNA. This cleavage event, located in the D6 expansion segment, is structurally stabilized by a network of interactions with surrounding ribosomal proteins, maintaining the integrity of the large subunit. Our comparative analysis revealed that this compensatory network preserves a canonical architecture nearly indistinguishable from intact mouse and human ribosomes. These findings resolve the structural basis of this unique cleavage, showing that it is a stable, integrated feature whose function is likely linked to more subtle regulatory mechanisms, rather than inducing major structural rearrangements.

biochemistry↗

bioRxiv2025

CleanFinder: Browser-Native Analysis of Editing Outcomes and Allelic States

Genome editing experiments routinely generate complex mixtures of alleles rather than single, predefined outcomes. Resolving these heterogeneous edits across diverse editing modalities, sequencing platforms, and multiplexed designs remains a persistent analytical challenge. To address this, we developed CleanFinder, a browser-native framework for genotyping genome editing outcomes using a constrained semi-global alignment strategy. Context-aware alignment modes support a broad spectrum of editing scenarios, including indels, base substitutions, and complex prime editing modifications, across nuclear and mitochondrial targets. Additional modules include an optional turbo mode for high-throughput heuristic alignment in exploratory workflows, and an allele-aware module that leverages heterozygous SNPs to detect allelic dropout. To evaluate scalability and practical performance, we applied CleanFinder to a primary small-molecule screen of 1,849 compounds in HEK293T cells. The software efficiently processed the dataset, enabling high-throughput comparison of editing outcomes and nomination of candidate compounds for follow-up analysis. Together, CleanFinder provides a flexible and scalable platform for genome editing analysis, enabling detailed genotyping and systematic comparison of editing outcomes across diverse edit types and genomic contexts.

genetics↗

bioRxiv2025

CSF single-cell RNA sequencing reveals clonally expanded CD4+ stem cell-like memory T cells in GAD65-antibody associated neurological syndromes

BackgroundGlutamic acid decarboxylase (GAD) antibody-associated autoimmune neurological syndromes (AINS) are a spectrum of autoimmune-mediated CNS disorders. While antibodies targeting the 65 kDa isoform of GAD are of high diagnostic value, T cell mediated cytotoxicity has been identified as a key component of disease pathogenesis. The precise pathophysiological mechanisms by which the disease is triggered and maintained, however, remain incompletely understood. MethodsWe performed single-cell transcriptome and immune repertoire sequencing (sc-seq) in CSF and blood of 8 anti-GAD65 AINS patients compared to 8 non-inflammatory controls. Monoclonal antibodies (mAbs) were synthesized from B cell receptor (BCR) data to evaluate the B cellular immune response. FindingsWe identified an increase and expansion of activated CD4+ stem cell-like memory T cells (TSCM) in the CSF of anti-GAD65 AINS patients. Expanded T cells showed increased expression of proinflammatory genes. The mAb analysis revealed a high frequency of GAD65-reactive BCRs in the CSF of anti-GAD65 AINS patients with increased somatic hypermutations compared to non-GAD-reactive BCRs and BCRs from controls. ConclusionsSc-seq identified clonally expanded CD4+ TSCM in the CSF of anti-GAD65 AINS patients harboring cytotoxic properties likely contributing to disease pathogenesis. GAD-reactive B cells circulate in the CSF of anti-GAD65 AINS patients further supporting the concept of an antigen-specific intrathecal immune response. Future studies need to clarify the actual pathogenicity of these immune cells and the link between T and B cellular immune mechanisms in the pathogenesis of anti-GAD65 AINS. FundingGerman Research Foundation (ERARE18-202 UltraAIE), German Federal Ministry of Education and Research (CONNECT GENERATE (2.0); 01GM1908A and 01GM2208A).

immunology↗

bioRxiv2025

Quantifying cortical maturational aspects during different vigilance states in preterm infants by advanced EEG analysis

Preterm birth is associated with numerous neurodevelopmental adverse outcomes, even in the absence of acquired lesions, as it occurs during a critical period of brain development. Clear organization of vigilance states can be recognized from 30-32 weeks postmenstrual age (PMA). In this study, we investigated whether spatial and temporal properties of neuronal oscillatory dynamics (i.e., phase synchronization, bistability, and cross-frequency coupling) during different vigilance states provide insights into cortical maturation in preterm infants born very low birth weight (VLBW) at low neurological risk and devoid of detectable brain lesions. We analyzed artifact-free video-polysomnographic data from 11 VLBW preterm infants (PMA at recording: 33.0 {+/-} 1.6 weeks) who underwent brain MRI at term-equivalent age. For each vigilance state, we computed the weighted Phase Lag Index (wPLI), Bistability Index (BiS), and Phase-Amplitude Coupling (PAC), both globally and across anterior and posterior regions, and examined their correlation with PMA at recording. wPLI, BiS, and PAC showed specific trends across vigilance states. BiS and PAC exhibited posterior-to-anterior differences and correlated with PMA. Our study suggests that these electrophysiological markers, particularly BiS and PAC, may serve as indices to monitor aspects of cortical maturation in VLBW at low neurological risk.

neuroscience↗

bioRxiv2025

Regulatory T cell stability determines the efficiency of bile duct regeneration during cholangitis.

Background and AimsCholangiopathies such as Primary Sclerosing Cholangitis (PSC) cause damage to the bile ducts and fibrosis with no effective cure. A reduction and impairment in the function of regulatory T cells (Tregs) occurs in PSC. Yet, it is currently unknown what consequence this has on bile duct regeneration. We investigate whether Tregs regulate bile duct regeneration and the dynamics of Tregs turnover during bile duct injury. Approach and ResultsWe used the transgenic Foxp3GFPDTR model to mimic reduced Tregs infiltration to the liver during bile duct injury and showed that reduced intrahepatic Tregs limits bile duct regeneration. Fate mapping of Tregs (Foxp3CreERTAi14) showed that Tregs acquire a pro-inflammatory phenotype within the inflammatory microenvironment, even after IL2 mediated Tregs expansion. Ox40L expression correlates with fibrosis in PSC patients. In the 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine (DDC)-diet mouse model of experimental cholangiopathy, combining IL2-complex administration and blocking Ox40L decreases periportal fibrosis level, increases Treg number and reduces the pro-inflammatory phenotype of Tregs. ConclusionsThese results indicate that Tregs mediate cholangiocyte response to biliary injury, and Tregs downregulate Foxp3 and acquire an inflammatory phenotype in an inflammatory microenvironment. Enhancing Treg numbers through IL2 complex administration and blocking Ox40 signalling simultaneously suppress the inflammatory phenotype of Tregs and reduces bile duct damage and fibrosis.

immunology↗

bioRxiv2025

Genome-wide DNA methylation study reveals specific signatures in the affected arterial tissue of giant cell arteritis patients

ObjectivesGiant cell arteritis (GCA) is a large-vessel vasculitis, potentially causing complications such as blindness and strokes. This study aims to gain insights into the pathogenesis of GCA by identifying specific DNA methylation signatures in the arterial tissue of patients with this vasculitis. MethodsDNA methylation profiling was analyzed in 79 temporal artery biopsy samples (69 patients with GCA and 10 controls) by performing an epigenome-wide association study (EWAS). Differential analysis was performed to identify differentially methylated positions (DMPs) and regions (DMRs). Lastly, we compared our findings with previous transcriptomics and epigenomics studies on GCA-affected arteries. ResultsEWAS identified 3,644 DMPs (FDR < 0.05, |{Delta}{beta}| > 0.3), indicating a profound alteration within GCA-affected arterial tissue. These DMPs were annotated to 1,517 potentially dysregulated genes. 282 additional genes were identified by annotation of significant DMRs. Pathway enrichment analysis revealed a significant alteration of inflammatory mechanisms, such as interleukins 2 and 7, as well as pathways related to vascular remodeling. Omics study comparison revealed 37 genes consistently affected across datasets, many of them linked to immune signaling and T cell regulation. Notably, markers of exhausted T cells, including SLAMF6 and HAVCR2, were present among them. ConclusionsOur study identified GCA-specific DNA methylation signatures in arterial tissue, revealing disrupted inflammatory and vascular pathways, and suggesting the involvement of exhausted T cells in this condition. These findings offer new insights into GCA pathogenesis and provide new potential targets for the treatment of this debilitating disease. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABS{square} Affected arteries by giant cell arteritis (GCA) exhibit unique epigenetic signatures, reflecting significant disruptions in gene regulation. However, small sample sizes have constrained the clinical translation and broader interpretation of these findings, leaving key gaps in understanding GCA pathogenesis and identifying therapeutic targets. What this study adds{square} This study provides the largest-to-date epigenome-wide DNA methylation profiling in GCA-affected arteries, uncovering thousands of epigenetic changes and revealing profound disruptions in inflammatory and vascular pathways, including IL-2, IL-7, and CXCR4 signaling. {square}We compiled 37 genes consistently affected across different omics datasets in GCA-affected arteries, providing a robust list of candidates for further research into GCA pathogenesis and treatment. {square}Our findings provide evidence of T cell exhaustion in GCA-affected arteries, supported by consistent changes in key markers such as SLAMF6 and HAVCR2 (TIM-3), suggesting a novel mechanism of immune dysregulation in GCA. How this study might affect research, practice or policy{square} This study nominates exhausted T cells, the NLRP3 inflammasome, and CXCR4 signaling as novel contributors in GCA pathogenesis, suggesting new therapeutic targets for further research in the treatment of this disease.

immunology↗

bioRxiv2025

Redox homeostasis in ferroptosis and aging: a causal role for fard-1 and dhs-25 in Caenorhabditis elegans

Aging is a natural process characterized by a progressive physiological decline that undermines health and well-being in the elderly population. It is widely accepted that an unbalanced redox state belongs to the hallmarks of aging, but its role as one of the main drivers of ferroptosis is quite recent. Ferroptosis is a form of iron-dependent cell death caused by massive phospholipid peroxidation. The excessive accumulation of intracellular reactive oxygen species and iron, as well as the failure of the main cellular antioxidant systems, cause ferroptotic cell death. While clear roles for ferroptosis in pathological conditions such as cancer or neurodegeneration have been described, its physiological roles and regulators are less clearly understood. Here, using Caenorhabditis elegans as a powerful model organism for aging studies, we uncover a role for ferroptosis in physiological aging mediated by disturbed redox homeostasis. We evaluated healthspan parameters in a C. elegans wild-type strain highlighting how several age-related features differentially decline during aging. A progressive loss of the capability to contrast external stressors, with an increase in hydroxyl radicals and a failure of the glutathione antioxidant system demonstrated the progressive disruption of redox homeostasis in older age. Moreover, we showed that selected genes involved in redox metabolism are downregulated with aging. Among them, mutant strains of the fatty acyl-CoA reductase, fard-1, and of the dehydrogenase, dhs-25, displayed higher sensitivity to a ferroptosis inducer, increased lipid peroxidation, anticipated drop in total glutathione and reduced lifespan. Accordingly, the expression of one of the closest mammalians dhs-25 homolog, the hydroxysteroid 17-Beta Dehydrogenase 8, was downregulated in cells which are more sensitive to ferroptosis. Our results clearly prove a causal role for ferroptosis in C. elegans aging driven by oxidative stress, unveiling novel genes involved in this connection that may constitute targets for possible interventions to improve healthy aging.

cell biology↗

bioRxiv2024

Global Biogeography of Prokaryotes in Mangrove Sediments: Spatial Patterns and Ecological Insights from 16S rDNA Metabarcoding

The global asymmetry in the floristic diversity distribution within mangrove ecosystems is well-documented. However, sediment microbial communities, which play crucial ecological roles, are often overlooked in mangrove biogeography studies. This study aimed to investigate the diversity, composition, and distribution of prokaryotic communities across distinct mangrove bioregions (12 countries and territories from the Caribbean bioregion, Southeast America, East Africa, Indo-Malaysia and Australasia). We conducted a meta-analysis by compiling 16S rDNA datasets from 17 previous studies (540 samples) over a six year period (2014-2020) and applied community ecology analyses combined with random forest models. Although no significant correlation was observed between tree richness and microbial diversity, a microbial hotspot was identified in the Indo-West Pacific region. Microbiota associated with different mangroves displayed opposite latitudinal diversity gradients and beta-dispersions. Distance-decay relationships were weak but statistically significant. Random forest models identified key microbial taxa, and the OTUs shared across all bioregions suggested the presence of a potential core mangrove microbiota. Taxonomic and statistical analyses underscored the great heterogeneity of microbiota composition, heavily influenced by the study (e.g., molecular and bioinformatic methodologies). Future research on mangrove microbiome would benefit from standardized sampling and sequencing methods. Despite current limitations, distance-decay relationship confirmed the influence of dispersal barriers for mangrove sediment microbiota. This study also suggests that environmental factors, rather than plant diversity alone, may play a prominent role in shaping the diversity, composition and distribution of prokaryotic communities in mangrove sediments.

microbiology↗

bioRxiv2024

Sociality shapes mitochondrial adaptations supporting hypoxia tolerance

Oxygen deprivation or hypoxia is poorly dealt with by most terrestrial species and often leads to permanent tissue damage and death. One prominent exception is the naked mole-rat (Heterocephalus glaber) which is remarkably adapted to withstand prolonged periods ([~]18 mins) of severe hypoxia, a trait likely driven by its crowded underground lifestyle. Other African mole-rat species are less social or entirely solitary like the Cape mole-rat (Georychus capensis). Here, we asked whether cellular and molecular adaptations to hypoxia map to social traits. We discovered that at the cellular level naked mole-rat fibroblasts survive >30 hours in 1% oxygen, while fibroblasts from terrestrial or non-social mole-rat species (human, mouse and Cape mole-rat) die rapidly under hypoxic conditions. We further show that naked mole-rat mitochondria have evolved morphological, functional and proteomic adaptations crucial for hypoxia resistance, remaining unaffected after long periods of severe hypoxia. We identify the mitochondrial protein Optic Atrophy 1 (OPA1) as a key player in naked mole-rat hypoxia resilience. Naked mole-rat mitochondria not only express more protective forms of OPA1, but also harbor a structurally unique isoform that likely protects cells from hypoxic damage. We show that evolutionary changes including the functionalization of a unique Opa1 exon support mitochondrial mediated cellular protection. Indeed, knockdown of OPA1 in naked mole-rat cells is sufficient to render them equally susceptible to hypoxia as human cells or cells from non-social African species. Our study demonstrates how molecular evolution drives unique adaptations that enable cells to achieve unprecedented resistance to hypoxic damage. We also show that molecular changes at the level of mitochondria are crucial in conferring hypoxia resistance. Our results thus chart a novel molecular path to understand how robust cellular hypoxia resistance can be achieved. Such knowledge may eventually inspire novel strategies to circumvent the consequences of hypoxic-damage in humans.

physiology↗

bioRxiv2024

Deep learning-driven neuromorphogenesis screenings identify repurposable drugs for mitochondrial disease

Mitochondrial disease encompasses untreatable conditions affecting tissues with high energy demands. A severe manifestation of mitochondrial disease is Leigh syndrome (Leigh), which causes defects in basal ganglia and midbrain regions, psychomotor regression, lactic acidosis, and early death. We previously generated isogenic pairs of Leigh cerebral organoids and uncovered defects in neuromorphogenesis. Here, we leveraged on this disease feature to devise drug discovery pipelines. We developed a deep learning algorithm tailored for cell type-specific drug repurposing to identify drugs capable of promoting neuronal commitment. In parallel, we performed a survival drug screen in yeast and validated the repurposable hits on branching capacity in Leigh neurons. The two approaches independently highlighted azole compounds, Talarozole and Sertaconazole, both of which lowered lactate release and improved neurogenesis and neurite organization in Leigh midbrain organoids. Hence, targeting neuromorphogenesis has led to identify potential new drugs for mitochondrial disease and could prove an effective strategy for further drug discovery.

neuroscience↗

bioRxiv2024

Reorganization of innate immune cell lipid profiles by bioinspired meroterpenoids to limit inflammation

Lipidomics-guided screening of unexplored chemical space in natural products provides access to small molecules capable of modifying cellular lipid profiles on a global scale. Here, we show that the meroterpenoid cyclosmenospongine from Spongia sp. shapes the lipid profile of immune cells, favoring anti-inflammatory and pro-resolving over pro-inflammatory lipid mediators. Structural variation revealed derivatives that inhibit leukotriene biosynthesis to varying extents while differentially upregulating pro-resolving lipid mediators, epoxyeicosatrienoic acids, endocannabinoids, and sphingosine-1-phosphate, along with other mediators, both in resting and activated innate immune cells in vitro and in self-resolving murine peritonitis in vivo. Mechanistically, meroterpenoids target 5-lipoxygenase or 5-lipoxygenase-activating protein, promote the translocation of 15-lipoxygenase-1 to cytoplasmatic sites, and inhibit monoacylglycerol lipase. They also redirect arachidonic acid (AA) from neutral lipids to specific phospholipids, while increasing the total concentration of free AA. Furthermore, meroterpenoids reprogram lipid metabolism in immune cells, decreasing the levels of neutral lipids, triacylglycerols, and cholesteryl esters. This shift correlates with a reduced capacity for leukotriene biosynthesis and is mimicked by the inhibition of sterol-O-acyltransferase and diacylglycerol acyltransferase-1/2. In conclusion, specific meroterpenoids exert anti-inflammatory effects by intervening in lipid mediator biosynthesis, prompting structure-controlled switches in lipid mediator classes, among others, through an unexpected link between lipogenesis and inflammation.

pharmacology and toxicology↗