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Replication of blood DNA methylomic signatures associated with cerebrospinal fluid levels of YKL-40 and NfL biomarkers
Get PDFRibo-Seq of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 via the auxin-inducible degron (AID) system
No full textUPF1 is a multi-domain RNA helicase that constantly monitors the transcriptome by non-specifically binding to mRNAs, dissociating from non-target transcripts, and initiating degradation on selected target RNAs via multiple proposed pathways such as nonsense-mediated decay (NMD). NMD is a translation-coupled mechanism that targets mRNAs harboring a premature stop codon (PTC) for degradation, thereby serving as a quality control and gene regulatory pathway ensuring transcriptome integrity. The UPF1 gene is essential in cultured human cells and previous studies relied mostly on RNA interference to downregulate UPF1. Here we established an auxin-inducible UPF1 degron system in the human colorectal adenocarcinoma cell line HCT116 by first inserting the auxin receptor F-box protein-encoding AtAFB2-mCherry in the AAVS1 locus, followed by tagging UPF1 at the N-terminus with an V5-AID-tag (AID = miniIAA7 = AtIAA7 amino acids 37–104). With this cell lines we performed Ribo-Seq (ribosome footprinting) to assess the effects of UPF1 depletion on translation. To this end, depletion of UPF1 was induced with 500 µM indole-3-acetic acid (IAA) for 12h. As control untreated cells were used
Cryo-mtscATAC-seq for single-cell mitochondrial DNA genotyping and clonal tracing in archived human tissues
Get PDFHigh-throughput clonal tracing of primary human samples relies on naturally occurring barcodes, such as somatic mitochondrial DNA (mtDNA) mutations detected via single-cell ATAC-seq (mtscATAC-seq). Fresh-frozen clinical specimens preserve tissue architecture but compromise cell integrity, thereby precluding their use in multiomic approaches such as mitochondrial genotyping at single-cell resolution. Here, we introduce Cryo-mtscATAC-seq, a broadly applicable method for diverse pathophysiological contexts to isolate nuclei with their associated mitochondria (“CryoCells”) from frozen samples for high-throughput clonal analysis. We applied Cryo-mtscATAC-seq to the neurodegenerated human brain, glioblastoma (GBM), pediatric neuroblastoma, and human aorta, and implemented mitobender, a computational tool to reduce ambient mtDNA in single-cell assays. Our approach revealed regional clonal gliogenesis and microglial expansions in amyotrophic lateral sclerosis (ALS), persistence of oligodendrocyte progenitor cell (OPC)-like clones in GBM recurrence, mtDNA depth heterogeneity after neuroblastoma chemotherapy, and oligoclonal proliferation of smooth muscle cells in human aorta. In conclusion, Cryo-mtscATAC-seq broadly extends mtDNA genotyping to archival frozen specimens across tissue types, opening new avenues for investigation of cell stateinformed clonality in human health and disease
Deep phenotyping of heart failure with preserved ejection fraction through multi-omics integration
Get PDFAIMS: Heart failure with preserved ejection fraction (HFpEF) has become the predominant form of heart failure and a leading cause of global cardiovascular morbidity and mortality. Due to its heterogeneous nature, HFpEF presents substantial challenges in diagnosis and management. Given the limited treatment options and lifestyle-associated comorbidities, early identification is crucial for establishing effective preventive strategies. Here, we introduce and validate a machine learning-based multi-omics approach that integrates clinical and molecular data to detect and characterize HFpEF. METHODS AND RESULTS: A supervised classifier was trained on a stratified subset of UK Biobank participants (n = 401 917) to identify phenotypic profiles associated with subsequent symptom-defined HFpEF during longitudinal follow-up. Model performance was validated in a non-overlapping hold-out subset from all 22 UK Biobank assessment centres (n = 100 446; 6726 HFpEF cases; 7394 with multi-omics data). The classifier demonstrated robust discriminatory performance, with a receiver operating characteristic area under the curve (ROC AUC) of 0.931 (95% confidence interval [CI] 0.930–0.931), a sensitivity of 0.857 (95% CI 0.855–0.860) and a specificity of 0.847 (95% CI 0.846–0.847). It identified individuals who subsequently developed HFpEF an average of 6.3 ± 3.9 years before symptom onset in asymptomatic individuals. Similarity network fusion (SNF) identified distinct subgroups, including a high-risk cluster characterized by elevated mortality and dysregulated inflammatory pathways, which was distinguishable with high accuracy (ROC AUC 0.988; 95% CI 0.985–0.990). CONCLUSIONS: We identified HFpEF phenotypes at an early stage, often several years before the onset of clinical symptoms, when the disease trajectory may still be amenable to modification. The molecular characterization provides novel insights into the underlying disease complexity and enables more refined risk stratification
A spinal circuit for skilled locomotion
Get PDFNeural circuits in the spinal cord have a critical role in integrating sensory information and descending commands to coordinate body movements. Defining the functional diversity of spinal neurons is therefore essential for understanding the mechanisms underlying motor control. In this study, by combining anatomical, molecular, and functional analyses in mice, we identified and characterized a subtype of spinal ascending neurons belonging to the V0 family. We found that V0g ascending neurons are integrated in lumbar sensorimotor circuits, and their function is specifically required for the execution of precise limb movements necessary for skilled locomotion. This work advances our understanding of the functional organization of V0 neurons and highlights a previously unappreciated role in adjusting body movements to the more demanding needs of skilled locomotor tasks
Identification of a spinal circuit for skilled locomotion (house mouse)
No full textNeural circuits in the spinal cord have a critical role in integrating sensory information and descending commands to coordinate body movements. Defining the functional diversity of spinal neurons is therefore essential for understanding the mechanisms underlying motor control. In this study, by combining anatomical, molecular, and functional analyses in mice, we identified and characterized a subtype of ascending spinal neurons belonging to the V0g family. We found that V0g ascending neurons are integrated in lumbar sensorimotor circuits and their function is specifically required for the execution of precise limb movements necessary for skilled locomotion. This work advances our understanding of the functional organization of V0 neurons and highlights a previously unappreciated role in adjusting body movements to the more demanding needs of skilled locomotor tasks Overall design: Rabies retrograde tracing was used to label with a fluorecent protein the nuclei of ascending and descending mouse spinal neurons reciprocally connecting the lumbar and cervical enlargements. Nuclei were isolated via fluorescence-activated nucleus sorting and analysed using sn-RNA-seq
The novel MuRF2 target SNX5 regulates PKA activity through stabilization of RI-α and controls myogenic differentiation
Get PDFBACKGROUND: Muscle RING finger (MuRF) proteins are striated muscle-specific E3 ubiquitin ligases essential for muscle homeostasis. Whereas MuRF1 is well known for its role in muscle atrophy, MuRF2 and MuRF3 contribute to microtubule stabilization, influencing muscle differentiation and function. Their cooperative functions in regulating myogenesis are unclear. This study aimed to identify novel MuRF2 and MuRF3 interaction partners and investigate their function in myogenic differentiation. METHODS: Interaction partners of MuRF2 and MuRF3 were identified using stable isotope labelling with amino acids in cell culture (SILAC), followed by affinity purification and quantitative mass spectrometry (AP-MS). Mechanistic analyses included co-immunoprecipitation, domain mapping, ubiquitination assays, protein stability measurements and endosome isolation. Myogenic differentiation was evaluated by immunocytochemistry, qRT-PCR and western blotting. Functional effects were assessed using CRISPR-Cas9-mediated knockout and siRNA silencing. RESULTS: We identified sorting nexin 5 (SNX5), a BAR and PX domain-containing retromer component involved in retrograde vesicular transport, as a novel MuRF2 and MuRF3 binding partner. Both coiled-coil domains of MuRF3 were required for SNX5 binding, and the BAR domain of SNX5 mediated interaction with MuRF2 and MuRF3. Immunofluorescence staining demonstrated MuRF3-SNX5 interaction and colocalization on early endosomes along microtubules in myocytes. MuRF2 promoted ubiquitination of SNX5 at lysines 290 and 324, leading to proteasomal degradation, whereas MuRF3 counteracted this effect. Mass spectrometry revealed the protein kinase A regulatory subunit (PKA-RI-α) as cargo of SNX5-coated early endosomes in myocytes. SNX5 knockout (SNX5-KO) reduced RI-α stability in myocytes, enhanced PKA activity and increased HDAC5 degradation via the autophagy-lysosomal pathway, leading to MEF2-mediated upregulation of myostatin. SNX5-KO impaired myogenesis, with significant reductions in myogenin/Myog (p < 0.005), myomaker/Mymk (p < 0.01), myomerger/Mymx (p < 0.005) and MyHC isoforms Myh2 and Myh4 (p < 0.01). Myostatin treatment mimicked the SNX5-KO phenotype, reducing fast-twitch MyHC isoforms Myh1, Myh2, Myh3 and Myh4 (p < 0.05 for all) and significantly lowering Myomaker, Myomerger and MyHC expression throughout differentiation (p < 0.05 for all). Morphologically, myostatin-treated cells were shorter and thinner and had fewer nuclei. Quantification showed reduced differentiation and fusion indices (p < 0.001) and fewer nuclei per myosin-positive cell (p < 0.01). CONCLUSIONS: MuRF2 and MuRF3 exert opposing effects on SNX5-mediated retrograde transport, influencing PKA signalling and myogenic differentiation. SNX5 stabilizes RI-α within early endosomes, facilitating ordered myogenic differentiation. Our findings expand the known functions of MuRF proteins beyond proteasomal degradation and identify SNX5 as a key regulator of PKA activity in muscle cells. These insights may provide novel therapeutic targets for muscle-related disorders
Spatial multiomics of acute myocardial infarction reveals immune cell infiltration through the endocardium
Get PDFMyocardial infarction (MI) continues to be a leading cause of death worldwide. Even though it is well established that the complex interplay between different cell types determines the overall healing response after MI, the precise changes in the tissue architecture are still poorly understood. In this study, we generated an integrative cellular map of the acute phase after murine MI using a combination of imaging-based transcriptomics (Molecular Cartography) and antibody-based highly multiplexed imaging (Sequential Immunofluorescence). This enabled us to evaluate cell type compositions and changes at subcellular resolution over time. We observed the recruitment of leukocytes to the infarcted heart through the endocardium and performed unbiased spatial proteomic analysis using Deep Visual Proteomics (DVP) to investigate the underlying mechanisms. DVP identified von Willebrand factor (vWF) as an upregulated mediator of inflammation 24 hours after MI, and functional blocking of vWF reduced the infiltration of C-C chemokine receptor 2 (Ccr2)-positive monocytes and worsened cardiac function after MI