Single cell transcriptome analysis of the THY-Tau22 mouse model of Alzheimer's disease reveals sex-dependent dysregulations.

Alzheimer's disease (AD) progression and pathology show pronounced sex differences, but the factors driving these remain poorly understood. To gain insights into early AD-associated molecular changes and their sex dependency for tau pathology in the cortex, we performed single-cell RNA-seq in the THY-Tau22 AD mouse model. By examining cell type-specific and cell type-agnostic AD-related gene activity changes and their sex-dimorphism for individual genes, pathways and cellular sub-networks, we identified both statistically significant alterations and interpreted the upstream mechanisms controlling them. Our results confirm several significant sex-dependent alterations in gene activity in the THY-Tau22 model mice compared to controls, with more pronounced alterations in females. Both changes shared across multiple cell types and cell type-specific changes were observed. The differential genes showed significant over-representation of known AD-relevant processes, such as pathways associated with neuronal differentiation, programmed cell death and inflammatory responses. Regulatory network analysis of these genes revealed upstream regulators that modulate many of the downstream targets with sex-dependent changes. Most key regulators have been previously implicated in AD, such as Egr1, Klf4, Chchd2, complement system genes, and myelin-associated glycoproteins. Comparing with similar data from the Tg2576 AD mouse model and human AD patients, we identified multiple genes with consistent, cell type-specific and sex-dependent alterations across all three datasets. These shared changes were particularly evident in the expression of myelin-associated genes such as Mbp and Plp1 in oligodendrocytes. In summary, we observed significant cell type-specific transcriptomic changes in the THY-Tau22 mouse model, with a strong over-representation of known AD-associated genes and processes. These include both sex-neutral and sex-specific patterns, characterized by consistent shifts in upstream master regulators and downstream target genes. Collectively, these findings provide insights into mechanisms influencing sex-specific susceptibility to AD and reveal key regulatory proteins that could be targeted for developing treatments addressing sex-dependent AD pathology.

Multiomics analysis identifies novel facilitators of human dopaminergic neuron differentiation.

Midbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinson's disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) human iPSC reporter line, we here generate time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicts novel regulators of mDAN differentiation and super-enhancers are used to identify key TFs. We find LBX1, NHLH1 and NR2F1/2 to promote mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. A more detailed investigation of TF targets reveals that NHLH1 promotes the induction of neuronal miR-124, LBX1 regulates cholesterol biosynthesis, and NR2F1/2 controls neuronal activity.

The use of a complex tetra-culture alveolar model to study the biological effects induced by gold nanoparticles with different physicochemical properties.

A substantial increase in engineered nanoparticles in consumer products has been observed, heightening human and environmental exposure. Inhalation represents the primary route of human exposure, necessitating a focus on lung toxicity studies. However, to avoid ethical concerns the use of in vitro models is an efficient alternative to in vivo models. This study utilized an in vitro human alveolar barrier model at air-liquid-interface with four cell lines, for evaluating the biological effects of different gold nanoparticles. Exposure to PEGylated gold nanospheres, nanorods, and nanostars did not significantly impact viability after 24 h, yet all AuNPs induced cytotoxicity in the form of membrane integrity impairment. Gold quantification revealed cellular uptake and transport. Transcriptomic analysis identified gene expression changes, particularly related to the enhancement of immune cells. Despite limited impact, distinct effects were observed, emphasizing the influence of nanoparticles physicochemical parameters while demonstrating the model's efficacy in investigating particle biological effects.

Forecasting the dynamics of a complex microbial community using integrated meta-omics.

Predicting the behaviour of complex microbial communities is challenging. However, this is essential for complex biotechnological processes such as those in biological wastewater treatment plants (BWWTPs), which require sustainable operation. Here we summarize 14 months of longitudinal meta-omics data from a BWWTP anaerobic tank into 17 temporal signals, explaining 91.1% of the temporal variance, and link those signals to ecological events within the community. We forecast the signals over the subsequent five years and use 21 extra samples collected at defined time intervals for testing and validation. Our forecasts are correct for six signals and hint on phenomena such as predation cycles. Using all the 17 forecasts and the environmental variables, we predict gene abundance and expression, with a coefficient of determination ≥0.87 for the subsequent three years. Our study demonstrates the ability to forecast the dynamics of open microbial ecosystems using interactions between community cycles and environmental parameters.

2-Hydroxyglutarate modulates histone methylation at specific loci and alters gene expression via Rph1 inhibition.

2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae, a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target.

Impaired neuron differentiation in GBA-associated Parkinson's disease is linked to cell cycle defects in organoids.

The mechanisms underlying Parkinson's disease (PD) etiology are only partially understood despite intensive research conducted in the field. Recent evidence suggests that early neurodevelopmental defects might play a role in cellular susceptibility to neurodegeneration. To study the early developmental contribution of GBA mutations in PD we used patient-derived iPSCs carrying a heterozygous N370S mutation in the GBA gene. Patient-specific midbrain organoids displayed GBA-PD relevant phenotypes such as reduction of GCase activity, autophagy impairment, and mitochondrial dysfunction. Genome-scale metabolic (GEM) modeling predicted changes in lipid metabolism which were validated with lipidomics analysis, showing significant differences in the lipidome of GBA-PD. In addition, patient-specific midbrain organoids exhibited a decrease in the number and complexity of dopaminergic neurons. This was accompanied by an increase in the neural progenitor population showing signs of oxidative stress-induced damage and premature cellular senescence. These results provide insights into how GBA mutations may lead to neurodevelopmental defects thereby predisposing to PD pathology.

Fiber deprivation and microbiome-borne curli shift gut bacterial populations and accelerate disease in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurological disorder characterized by motor dysfunction, dopaminergic neuron loss, and alpha-synuclein (αSyn) inclusions. Many PD risk factors are known, but those affecting disease progression are not. Lifestyle and microbial dysbiosis are candidates in this context. Diet-driven gut dysbiosis and reduced barrier function may increase exposure of enteric neurons to toxins. Here, we study whether fiber deprivation and exposure to bacterial curli, a protein cross-seeding with αSyn, individually or together, exacerbate disease in the enteric and central nervous systems of a transgenic PD mouse model. We analyze the gut microbiome, motor behavior, and gastrointestinal and brain pathologies. We find that diet and bacterial curli alter the microbiome and exacerbate motor performance, as well as intestinal and brain pathologies, but to different extents. Our results shed important insights on how diet and microbiome-borne insults modulate PD progression via the gut-brain axis and have implications for lifestyle management of PD.

Formate promotes invasion and metastasis in reliance on lipid metabolism.

Metabolic rewiring is essential for cancer onset and progression. We previously showed that one-carbon metabolism-dependent formate production often exceeds the anabolic demand of cancer cells, resulting in formate overflow. Furthermore, we showed that increased extracellular formate concentrations promote the in vitro invasiveness of glioblastoma cells. Here, we substantiate these initial observations with ex vivo and in vivo experiments. We also show that exposure to exogeneous formate can prime cancer cells toward a pro-invasive phenotype leading to increased metastasis formation in vivo. Our results suggest that the increased local formate concentration within the tumor microenvironment can be one factor to promote metastases. Additionally, we describe a mechanistic interplay between formate-dependent increased invasiveness and adaptations of lipid metabolism and matrix metalloproteinase activity. Our findings consolidate the role of formate as pro-invasive metabolite and warrant further research to better understand the interplay between formate and lipid metabolism.

Metabolic modelling-based in silico drug target prediction identifies six novel repurposable drugs for melanoma.

Despite high initial response rates to targeted kinase inhibitors, the majority of patients suffering from metastatic melanoma present with high relapse rates, demanding for alternative therapeutic options. We have previously developed a drug repurposing workflow to identify metabolic drug targets that, if depleted, inhibit the growth of cancer cells without harming healthy tissues. In the current study, we have applied a refined version of the workflow to specifically predict both, common essential genes across various cancer types, and melanoma-specific essential genes that could potentially be used as drug targets for melanoma treatment. The in silico single gene deletion step was adapted to simulate the knock-out of all targets of a drug on an objective function such as growth or energy balance. Based on publicly available, and in-house, large-scale transcriptomic data metabolic models for melanoma were reconstructed enabling the prediction of 28 candidate drugs and estimating their respective efficacy. Twelve highly efficacious drugs with low half-maximal inhibitory concentration values for the treatment of other cancers, which are not yet approved for melanoma treatment, were used for in vitro validation using melanoma cell lines. Combination of the top 4 out of 6 promising candidate drugs with BRAF or MEK inhibitors, partially showed synergistic growth inhibition compared to individual BRAF/MEK inhibition. Hence, the repurposing of drugs may enable an increase in therapeutic options e.g., for non-responders or upon acquired resistance to conventional melanoma treatments.

Transcriptional and Chromatin Accessibility Profiling of Neural Stem Cells Differentiating into Astrocytes Reveal Dynamic Signatures Affected under Inflammatory Conditions.

Astrocytes arise from multipotent neural stem cells (NSCs) and represent the most abundant cell type of the central nervous system (CNS), playing key roles in the developing and adult brain. Since the differentiation of NSCs towards a gliogenic fate is a precisely timed and regulated process, its perturbation gives rise to dysfunctional astrocytic phenotypes. Inflammation, which often underlies neurological disorders, including neurodevelopmental disorders and brain tumors, disrupts the accurate developmental process of NSCs. However, the specific consequences of an inflammatory environment on the epigenetic and transcriptional programs underlying NSCs' differentiation into astrocytes is unexplored. Here, we address this gap by profiling in mice glial precursors from neural tissue derived from early embryonic stages along their astrocytic differentiation trajectory in the presence or absence of tumor necrosis factor (TNF), a master pro-inflammatory cytokine. By using a combination of RNA- and ATAC-sequencing approaches, together with footprint and integrated gene regulatory network analyses, we here identify key differences during the differentiation of NSCs into astrocytes under physiological and inflammatory settings. In agreement with its role to turn cells resistant to inflammatory challenges, we detect Nrf2 as a master transcription factor supporting the astrocytic differentiation under TNF exposure. Further, under these conditions, we unravel additional transcriptional regulatory hubs, including Stat3, Smad3, Cebpb, and Nfkb2, highlighting the interplay among pathways underlying physiological astrocytic developmental processes and those involved in inflammatory responses, resulting in discrete astrocytic phenotypes. Overall, our study reports key transcriptional and epigenetic changes leading to the identification of molecular regulators of astrocytic differentiation. Furthermore, our analyses provide a valuable resource for understanding inflammation-induced astrocytic phenotypes that might contribute to the development and progression of CNS disorders with an inflammatory component.

Altered infective competence of the human gut microbiome in COVID-19.

BACKGROUND: Infections with SARS-CoV-2 have a pronounced impact on the gastrointestinal tract and its resident microbiome. Clear differences between severe cases of infection and healthy individuals have been reported, including the loss of commensal taxa. We aimed to understand if microbiome alterations including functional shifts are unique to severe cases or a common effect of COVID-19. We used high-resolution systematic multi-omic analyses to profile the gut microbiome in asymptomatic-to-moderate COVID-19 individuals compared to a control group. RESULTS: We found a striking increase in the overall abundance and expression of both virulence factors and antimicrobial resistance genes in COVID-19. Importantly, these genes are encoded and expressed by commensal taxa from families such as Acidaminococcaceae and Erysipelatoclostridiaceae, which we found to be enriched in COVID-19-positive individuals. We also found an enrichment in the expression of a betaherpesvirus and rotavirus C genes in COVID-19-positive individuals compared to healthy controls. CONCLUSIONS: Our analyses identified an altered and increased infective competence of the gut microbiome in COVID-19 patients. Video Abstract.

Systems level analysis of sex-dependent gene expression changes in Parkinson's disease.

Parkinson's disease (PD) is a heterogeneous disorder, and among the factors which influence the symptom profile, biological sex has been reported to play a significant role. While males have a higher age-adjusted disease incidence and are more frequently affected by muscle rigidity, females present more often with disabling tremors. The molecular mechanisms involved in these differences are still largely unknown, and an improved understanding of the relevant factors may open new avenues for pharmacological disease modification. To help address this challenge, we conducted a meta-analysis of disease-associated molecular sex differences in brain transcriptomics data from case/control studies. Both sex-specific (alteration in only one sex) and sex-dimorphic changes (changes in both sexes, but with opposite direction) were identified. Using further systems level pathway and network analyses, coordinated sex-related alterations were studied. These analyses revealed significant disease-associated sex differences in mitochondrial pathways and highlight specific regulatory factors whose activity changes can explain downstream network alterations, propagated through gene regulatory cascades. Single-cell expression data analyses confirmed the main pathway-level changes observed in bulk transcriptomics data. Overall, our analyses revealed significant sex disparities in PD-associated transcriptomic changes, resulting in coordinated modulations of molecular processes. Among the regulatory factors involved, NR4A2 has already been reported to harbor rare mutations in familial PD and its pharmacological activation confers neuroprotective effects in toxin-induced models of Parkinsonism. Our observations suggest that NR4A2 may warrant further research as a potential adjuvant therapeutic target to address a subset of pathological molecular features of PD that display sex-associated profiles.

RvD1n-3 DPA Downregulates the Transcription of Pro-Inflammatory Genes in Oral Epithelial Cells and Reverses Nuclear Translocation of Transcription Factor p65 after TNF-α Stimulation.

Specialized pro-resolving mediators (SPMs) are multifunctional lipid mediators that participate in the resolution of inflammation. We have recently described that oral epithelial cells (OECs) express receptors of the SPM resolvin RvD1n-3 DPA and that cultured OECs respond to RvD1n-3 DPA addition by intracellular calcium release, nuclear receptor translocation and transcription of genes coding for antimicrobial peptides. The aim of the present study was to assess the functional outcome of RvD1n-3 DPA-signaling in OECs under inflammatory conditions. To this end, we performed transcriptomic analyses of TNF-α-stimulated cells that were subsequently treated with RvD1n-3 DPA and found significant downregulation of pro-inflammatory nuclear factor kappa B (NF-κB) target genes. Further bioinformatics analyses showed that RvD1n-3 DPA inhibited the expression of several genes involved in the NF-κB activation pathway. Confocal microscopy revealed that addition of RvD1n-3 DPA to OECs reversed TNF-α-induced nuclear translocation of NF-κB p65. Co-treatment of the cells with the exportin 1 inhibitor leptomycin B indicated that RvD1n-3 DPA increases nuclear export of p65. Taken together, our observations suggest that SPMs also have the potential to be used as a therapeutic aid when inflammation is established.

Single-Cell Transcriptional Profiling and Gene Regulatory Network Modeling in Tg2576 Mice Reveal Gender-Dependent Molecular Features Preceding Alzheimer-Like Pathologies.

Alzheimer's disease (AD) onset and progression is influenced by a complex interplay of several environmental and genetic factors, one of them gender. Pronounced gender differences have been observed both in the relative risk of developing AD and in clinical disease manifestations. A molecular level understanding of these gender disparities is still missing, but could provide important clues on cellular mechanisms modulating the disease and reveal new targets for gender-oriented disease-modifying precision therapies. We therefore present here a comprehensive single-cell analysis of disease-associated molecular gender differences in transcriptomics data from the neocortex, one of the brain regions most susceptible to AD, in one of the most widely used AD mouse models, the Tg2576 model. Cortical areas are also most commonly used in studies of post-mortem AD brains. To identify disease-linked molecular processes that occur before the onset of detectable neuropathology, we focused our analyses on an age with no detectable plaques and microgliosis. Cell-type specific alterations were investigated at the level of individual genes, pathways, and gene regulatory networks. The number of differentially expressed genes (DEGs) was not large enough to build context-specific gene regulatory networks for each individual cell type, and thus, we focused on the study of cell types with dominant changes and included analyses of changes across the combination of cell types. We observed significant disease-associated gender differences in cellular processes related to synapse organization and reactive oxygen species metabolism, and identified a limited set of transcription factors, including Egr1 and Klf6, as key regulators of many of the disease-associated and gender-dependent gene expression changes in the model. Overall, our analyses revealed significant cell-type specific gene expression changes in individual genes, pathways and sub-networks, including gender-specific and gender-dimorphic changes in both upstream transcription factors and their downstream targets, in the Tg2576 AD model before the onset of overt disease. This opens a window into molecular events that could determine gender-susceptibility to AD, and uncovers tractable target candidates for potential gender-specific precision medicine for AD.

Elucidating tumour-associated microglia/macrophage diversity along glioblastoma progression and under ACOD1 deficiency.

In glioblastoma (GBM), tumour-associated microglia/macrophages (TAMs) represent the major cell type of the stromal compartment and contribute to tumour immune escape mechanisms. Thus, targeting TAMs is emerging as a promising strategy for immunotherapy. However, TAM heterogeneity and metabolic adaptation along GBM progression represent critical features for the design of effective TAM-targeted therapies. Here, we comprehensively study the cellular and molecular changes of TAMs in the GL261 GBM mouse model, combining single-cell RNA-sequencing with flow cytometry and immunohistological analyses along GBM progression and in the absence of Acod1 (also known as Irg1), a key gene involved in the metabolic reprogramming of macrophages towards an anti-inflammatory phenotype. Similarly to patients, we identify distinct TAM profiles, mainly based on their ontogeny, that reiterate the idea that microglia- and macrophage-like cells show key transcriptional differences and dynamically adapt along GBM stages. Notably, we uncover decreased antigen-presenting cell features and immune reactivity in TAMs along tumour progression that are instead enhanced in Acod1-deficient mice. Overall, our results provide insight into TAM heterogeneity and highlight a novel role for Acod1 in TAM adaptation during GBM progression.

Evolution of the murine gut resistome following broad-spectrum antibiotic treatment.

The emergence and spread of antimicrobial resistance (AMR) represent an ever-growing healthcare challenge worldwide. Nevertheless, the mechanisms and timescales shaping this resistome remain elusive. Using an antibiotic cocktail administered to a murine model along with a longitudinal sampling strategy, we identify the mechanisms by which gut commensals acquire antimicrobial resistance genes (ARGs) after a single antibiotic course. While most of the resident bacterial populations are depleted due to the treatment, Akkermansia muciniphila and members of the Enterobacteriaceae, Enterococcaceae, and Lactobacillaceae families acquire resistance and remain recalcitrant. We identify specific genes conferring resistance against the antibiotics in the corresponding metagenome-assembled genomes (MAGs) and trace their origins within each genome. Here we show that, while mobile genetic elements (MGEs), including bacteriophages and plasmids, contribute to the spread of ARGs, integrons represent key factors mediating AMR in the antibiotic-treated mice. Our findings suggest that a single course of antibiotics alone may act as the selective sweep driving ARG acquisition and incidence in gut commensals over a single mammalian lifespan.

Focal adhesion kinase plays a dual role in TRAIL resistance and metastatic outgrowth of malignant melanoma.

Despite remarkable advances in therapeutic interventions, malignant melanoma (MM) remains a life-threating disease. Following high initial response rates to targeted kinase-inhibition metastases quickly acquire resistance and present with enhanced tumor progression and invasion, demanding alternative treatment options. We show 2nd generation hexameric TRAIL-receptor-agonist IZI1551 (IZI) to effectively induce apoptosis in MM cells irrespective of the intrinsic BRAF/NRAS mutation status. Conditioning to the EC50 dose of IZI converted the phenotype of IZI-sensitive parental MM cells into a fast proliferating and invasive, IZI-resistant metastasis. Mechanistically, we identified focal adhesion kinase (FAK) to play a dual role in phenotype-switching. In the cytosol, activated FAK triggers survival pathways in a PI3K- and MAPK-dependent manner. In the nucleus, the FERM domain of FAK prevents activation of wtp53, as being expressed in the majority of MM, and consequently intrinsic apoptosis. Caspase-8-mediated cleavage of FAK as well as FAK knockdown, and pharmacological inhibition, respectively, reverted the metastatic phenotype-switch and restored IZI responsiveness. FAK inhibition also re-sensitized MM cells isolated from patient metastasis that had relapsed from targeted kinase inhibition to cell death, irrespective of the intrinsic BRAF/NRAS mutation status. Hence, FAK-inhibition alone or in combination with 2nd generation TRAIL-receptor agonists may be recommended for treatment of initially resistant and relapsed MM, respectively.

Critical Assessment of MetaProteome Investigation (CAMPI): a multi-laboratory comparison of established workflows.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

Functional meta-omics provide critical insights into long- and short-read assemblies.

Real-world evaluations of metagenomic reconstructions are challenged by distinguishing reconstruction artifacts from genes and proteins present in situ. Here, we evaluate short-read-only, long-read-only and hybrid assembly approaches on four different metagenomic samples of varying complexity. We demonstrate how different assembly approaches affect gene and protein inference, which is particularly relevant for downstream functional analyses. For a human gut microbiome sample, we use complementary metatranscriptomic and metaproteomic data to assess the metagenomic data-based protein predictions. Our findings pave the way for critical assessments of metagenomic reconstructions. We propose a reference-independent solution, which exploits the synergistic effects of multi-omic data integration for the in situ study of microbiomes using long-read sequencing data.

Single-Cell Transcriptomics and In Situ Morphological Analyses Reveal Microglia Heterogeneity Across the Nigrostriatal Pathway.

Microglia are the resident immune effector cells of the central nervous system (CNS) rapidly reacting to various pathological stimuli to maintain CNS homeostasis. However, microglial reactions in the CNS may also worsen neurological disorders. Hence, the phenotypic analysis of microglia in healthy tissue may identify specific poised subsets ultimately supporting or harming the neuronal network. This is all the more important for the understanding of CNS disorders exhibiting regional-specific and cellular pathological hallmarks, such as many neurodegenerative disorders, including Parkinson's disease (PD). In this context, we aimed to address the heterogeneity of microglial cells in susceptible brain regions for PD, such as the nigrostriatal pathway. Here, we combined single-cell RNA-sequencing with immunofluorescence analyses of the murine nigrostriatal pathway, the most affected brain region in PD. We uncovered a microglia subset, mainly present in the midbrain, displaying an intrinsic transcriptional immune alerted signature sharing features of inflammation-induced microglia. Further, an in situ morphological screening of inferred cellular diversity showed a decreased microglia complexity in the midbrain when compared to striatum. Our study provides a resource for the identification of specific microglia phenotypes within the nigrostriatal pathway, which may be relevant in PD.